Femtosecond laser direct writing technology exhibits a variety of significant advantages compared to traditional micromachining technologies, such as the high processing resolution beyond the optical diffraction limit, a wide range of processing materials, and “real” three-dimensional structures fabrication. Due to these unique advantages, it is typically used for fabricating the micro-optical devices, biomedical devices, miniaturized electromechanical systems, etc. In aspect of the fabrication resolution, it usually depends on the use of the objective lens with the high numerical aperture in the femtosecond laser fabrication setup. Caused by the small depth of field of the high numerical aperture, it is easy to produce the out of focus effect, which leads to the error of the fabricated microstructures. Thus, it is vital and significant for quickly and accurately locating the laser focus on the surface of the sample during the micro- and nanostructures fabrication process.To solve the problem of quantitatively achieving the auto-focus function in femtosecond laser three-dimensional fabrication, this paper proposes a new type of automatic focusing method that it includes the computed light field distribution and the image processing algorithms. First of all, in the situation of tightly focusing of the high numerical aperture objective lens, the laser light field distribution function in the multilayer materials after passing the objective lens are derived according to the Richards-Wolf vector diffraction theory. Furthermore, the light field distribution results at the different positions of the medium can be solved by the numerical simulation of the laser light field distribution function. In the meantime, the laser beam diameter as a function of the position in the multilayer mediums can be also theoretically obtained. Secondly, a series of the laser beam images are captured in the interval of 2 μm by moving the laser focus from top to bottom of the multilayer mediums. And the laser beam images are used for the image processing to obtain the laser beam diameter, that specific image processing algorithms are adopted here, including region of interest, open and closed operations, maximum interclass difference method, corrosion and expansion algorithm, Canny edge extraction processing algorithm processing. Next, the laser beam images after the image processing are further processed with the least squares circle fitting method to obtain the laser beam diameter at different positions in the multilayer medium and the curve of the laser beam diameter varying with different positions are also obtained. Finally, the mathematical model of the laser beam diameter and out-of-focus amount is obtained by comparing with the simulated and experimental results. The correction proportion is calculated based on the mathematical model, which is regarded as the amendatory curve. The amendatory model is used to realize the autofocus of the femtosecond laser fabrication system, which is achieved by capturing the real-time laser beam image and comparing with the curve model, then adjusting the actual position to the laser focal position. Finally, an automatic focusing method is successfully obtained based on the calculation of optical field distribution and image processing algorithm. Besides, the ultrashort pulse duration of femtosecond laser means that it has a range width of spectrums. In our experiments, the femtosecond laser from the spectrum-physics company with the central wavelength of ~800 nm and the width of spectrum of ~60 nm is used. Furthermore, the laser light field distribution is studies by considering the influence of different wavelengths, and the ratio coefficient after considering dispersion are theoretically calculated in the paper. The simulation results indicates that with the spectral width increasing, the simulation data are closer to the experimental data, that is, the calculated error ratio coefficient shows an increasing, then a decreasing trend.This kind of autofocus method can obtain the high accuracy out-of-focus amount in real-time during femtosecond laser fabrication process, thereby it can finely tune the focus position after comparing with the curve model. This method provides a solution for the microstructure fabrication of the non-planar and curved substrates and can promote the application field of femtosecond laser fabrication technology.