【Aim】 The insect fusion protein Cry1Ab/Vip3Da of transgenic soybean CAL16 has shown good resistance against soybean lepidopteran pests; however, a qualitative detection method for insect-resistant soybean CAL16 and its derivatives has not yet been established. The purpose of this study was to design a specific qualitative polymerase chain reaction (PCR) detection primer for CAL16 transformants based on the insertion position of exogenous fragments to fill the gap in the standard. 【Method】 Based on the insertion position and vector information published in the technical data, multiple pairs of primers were designed at the left boundary (LB terminus) and right boundary (RB terminus). The primers were subjected to specificity testing, amplification reaction optimization, sequencing verification, robustness testing, sensitivity testing, and detection limit testing. 【Result】 The data showed that the primer combination CAL16-F/R had good specificity and robustness and that the detection limit was 0.10%, which met the requirements of relevant transgenic molecular testing standards. Eight genetically modified organism (GMO) safety testing institutions in China were able to replicate the test results. Therefore, this method can be used in the testing industry. 【Conclusion】 The establishment of a qualitative PCR detection method for CAL16 in transgenic insect-resistant soybean provides technical support for the safety supervision of this transformant and its derivatives in China.