A novel technique of one-shot in-line digital holography based on double wavelength illumination was proposed. Two wavelength beams in color of red,green or blue (RGB) are simultaneously used to illuminate the object and produce the in-line digital hologram (DH). After recording the hologram by a color CCD,two in-line DHs recorded with different wavelengths can be obtained by separating the color image into RGB channels. The angular spectrum theory of diffraction was applied in the analysis of the proposed technique. On weakening the object wave in comparison to the reference wave,the summation of the diffraction object wave and its conjugation can be approximately obtained by logarithmizing the in-line DH. The approximating error lies on the object and reference wave ratio. Then the object image can be reconstructed from the two in-line DHs of different wavelengths by applying the transfer function. The reconstruction method was presented. Computer simulations and simple experiment were given to demonstrate the technique. The peak signal noise ratio (PSNR) of the reconstructed image is about 14.2 dB. Which shows this method could erase the interference of twin diffraction image. Finally,the noise sources of the reconstructed images were simply analyzed. This technique can be used for real time imaging and dynamic measuring based in-line digital holography.