C-peptide level is recognized as an important indicator of diabetes diagnosis. A sensitive and specific double-antibody sandwich enzyme-linked immunosorbent assay for the detection of C-peptide based on double antibody sandwich method was studied in this paper. The rabbit and hen were innunized with PLL-C-peptide and BSA-C-peptide respectively to obtain specific Yolk antibody (IgY) and polyclonal antibody used to construct the sandwich ELISA for the measurement of C-peptide. The limit of detection was 0.51 μg/mL and the half maximal inhibitory concentration (IC50) was 3.26 μg/mL. The method developed in the study showed no evident cross-reactivity with other similar analogs. The detection standard curve of C-peptide exhibited a good linearity (R2 = 0.9896, n = 15). 17 types of the urine of diabetes patients on c-peptide levels compared with the hospital type of diabetes information, with a conclusion of a high consistent rate. Therefore, the methods could be selectively used for rapid screening of C-peptide in human urine, and the type of diabetes has some referential significance.