In noninvasive biochemical detection, the differential spectrum method based on the change in blood volume can eliminate the interference of human tissue background in theory, and obtain effective spectrum information of blood. In order to demonstrate the effectiveness of the differential spectrum method, simulated experiment was designed. Biological molecules solutions were used for simulating serum sample, filters with different absorption characteristic were used for simulating interference of tissue background, and an adjustable path-length cell was used for simulating blood volume change. Model accuracies of pre- and post-treatment with differential spectrum method were compared. Thus treated, the root mean square error of cross validation (RMSECV) reduced from 437 to 301 mg·dL-1. The experimental results indicate that using the differential spectrum method can effectively restrain the interference of tissue background, and greatly improve the prediction precision of calibration model.