The traditional microscope is not suitable to observe the schistosoma cercariae which usually floating on the water surface, because it needs an optical focusing process and the wobble or fluctuation of the water surface is difficult to avoid. The basic concept and procedure of digital in-line holography based on convolution are shown mainly for schistosoma cercariae detection. An auto-focus algorithm based on wavelet transform was improved. The max amplitude of high-frequency coefficients in the focal windows was used to evaluate the definition of the reconstructed image instead of the sum of high-frequency coefficients. The evaluation function was maximal only when the reconstructed distance equal to the recording distance in the simulated experiment, which shows the accuracy of the improved algorithm. The experimental device of digital in-line holography was designed for schistosoma cercariae detection. The experimental results show that the auto-focusing method based on wavelet transform can be used in digital holography, and the digital in-line holography can be used to detect schistosoma cercariae when the water surface is fluctuant. The reconstructed image resolution is equivalent to the resolution of digital microscope with 1X micro lens, and the bifurcated tail of schistosoma cercariae can be observed clearly.