Laser Induced Fluorescence Detection (LIFD) technology is one of the most sensitive means for the microfluidic chip detection in which application field it is important to achieve accurate focus due to the small size of microfluidic chip. A novel LIFD based on liquid lens is proposed to obtain the optimal excitation light and the induced fluorescence collection, which is capable of adjusting focus position in three-dimension automatically according to the information provided by the images. The autofocus algorithm for LIFD is accomplished with the combination of SUSAN operator and the mountain climbing control algorithm. A tunable liquid lens based on LIFD experimental prototype is set up to testify the proposed system and algorithms. Experimental results show that the system can achieve the autofocus of microfluidic chip. The repeatability experimental results indicate that the evaluation of the last picture in each single experiment has a steady feature with a fluctuation of 0.5% and coefficient of variation of 1.5‰. It provides a solution to improving the detection accuracy and a design flexibility of microfluidic chip.