To simultaneously detecting the C-peptide and insulin in human serum, a dual-labeling time-resolved fluoroimmunoassay(TRFIA) for detection of C-peptide and insulin was preliminary founded. Two capture monoclonal antibodies were mixed together and then coated in human serum. Two counterpart tracer monoclonal antibodies, were labeled with Eu³⁺ (anti-Insulin) and Sm³⁺ (anti-C-peptide) chelates, respectively. The double antibody sandwich in one step method was used to develop the TRFIA for simultaneously detection of the C-peptide and insulin in human serum. These results showed that sensitivity of C peptide analysis was up to 0.2 μg/L, linear range is 0.5～22 μg/L, the recovery percent is 99.6%, The intra- and inter-assay coefficient of variation were 4.6%～6.0% and 5.1%～7.6%, respectively; sensitivity of insulin analysis was up to 0.8 mU/L, linear range is 3.6～180 mU/L, the recovery percent is 99.4%, the intra- and inter-assay coefficient of variation were 3.7%～6.0% and 5.1%～8.0%, respectively. Moreover, 200 samples were tested by the self-made and commercially available C-peptide and insulin kit (PerkinElmer) kits at the same time. The correlation coefficient was 0.98 and 0.99, respectively. In conclusion, the self-made dual-labeling time-resolved fluoroimmunoassay for simultaneously detection of C-peptide and insulin might be a simple, sensitive and rapid method for rapid diagnosis for the diabetes caused by the lack of insulin secretion, it would also provide an alternative route for serology high-screening of the samples for C-peptide and insulin in clinical application.