Abstract: To explore the endocrine disrupting gene markers of invertebrates based on the insulin signal pathway, this study selected Chironomus tentans as the model organism, identified the key genes in the insulin signal pathway on the basis of transcriptome sequencing, and carried out the full-length amplification of cDNA and the multi-species comparison of amino acid sequences for the upstream genes such as InR, Pdk, Akt and FoxO. Based on the cDNA full-length sequence, the InR, Pdk, Akt and FoxO genes of Chironomus tentans can encode protein sequences containing 1 436, 522, 520 and 470 amino acids, respectively. The sequence homology comparison and phylogenetic tree analysis results verified its evolutionary conservatism. The qRT-PCR results of the relative expression of genes in Ⅰ-Ⅳ instar larvae, pupae, female adults and male adults show that the expression trend of InR, Pdk, Akt and FoxO in C. tentans was similar. The peaks of gene expression in I and IV instar indicate that insulin signaling induces faster growth and development rates in I and IV instar larvae. The expression of InR, Pdk, Akt, and FoxO in female adults was significantly higher than that in males, which may be related to the involvement of insulin signaling in the development of the female reproductive system.