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The traditional focusing function and the experimental parameters in digital in-line holography for particle field detection is not suitable for schistosoma cercariae detection. The basic concept and procedure of digital in-line holography were shown mainly for schistosoma cercariae detection. A digital focusing method based on Laplacian operator was presented. The feasibility of schistosoma cercariae detection with digital in-line holography was confirmed through computer simulation. The results show Laplacian operator could be used as the automatic digital focusing function in digital holographic reconstruction. Furthermore, the range of recording distance was given by formula Δx·L/λ<;60;z0<;L2/(160·λ) in digital in-line holography for schistosoma cercariae detection, and the best recording distance wasΔx·L/λ.