Abstract: 【Objective】The purpose of the study was to screen the suitable reference gene and the optimal number of combinations for Zeugodacus cucurbitae(Coquillett)insecticides resistance and physiological molecular mechanism study,thus providing a foundation for the study of Z. cucurbitae insecticide resistance mechanism.【Method】Sensitive and abamectin resistant strains of Z. cucurbitae in different development periods and different tissue samples were used as test materials. The eight candidate reference genes were expressed by real-time fluorescence quantitative PCR. The expression stability of reference genes was analyzed by comprehensive assessment using geNorm,NormFinder,BestKeeper and RefFinder. The most stably expressed reference genes were selected and the optimal number of combinations was clarified.【Result】The results of geNorm analysis indicated that the expression stability rank of the reference genes treated in different development periods was RPS13=RpL32(0.269)>TUBE(0.623)>TBP(0.702)>β-tubulin(1.069)>G6PDH(1.366)> Actin3(1.596)>Actin2(1.923). The expression stability rank of the reference genes treated in different tissues was RpL32=RPS13(0.132)>TBP(0.345)>TUBE(0.442)>β-tubulin(0.552)>G6PDH(0.780)>Actin2(1.101)>Actin3(1.417),and the optimal number of reference gene was 2. The results of NormFinder analysis indicated that the expression stability rank of the reference genes treated in different development periods was RpL32(0.399)>RPS13(0.446)>TUBE(0.738)>G6PDH (0.912)>TBP(0.924)>β-tubulin(1.086)>Actin3(1.092)>Actin2(1.866). The expression stability rank of the reference genes treated in different tissues was RpL32(0.090)>RPS13(0.117)>TBP(0.389)>TUBE(0.445)>β-tubulin(0.705)> G6PDH(0.877)>Actin2(1.009)>Actin3(1.567). The results of BestKeeper analysis indicated that the expression stability rank of the reference genes treated in different development periods was TUBE>TBP>RPS13>RpL32>β-tubulin>G6PDH> Actin3>Actin2. The expression stability rank of the reference genes treated in different tissues was β-tubulin>TBP>TUBE> RPS13>RpL32>G6PDH>Actin2>Actin3. The results of RefFinder analysis indicated that the expression stability rank of the reference genes treated in different development period was RpL32>RPS13>TUBE>TBP>G6PDH>β-tubulin>Actin3> Actin2. The expression stability rank of the reference genes treated in different tissue was RPS13>RpL32>TBP>β-tubulin> TUBE>G6PDH>Actin2>Actin3.【Conclusion】RPS13 and RpL32 can be combined as the reference gene for the study of different development periods,different tissues,abamectin resistance molecular mechanism and physiological molecular mechanism in Z. cucurbitae.