Spotted knifejaw (Oplegnathus punctatus) is a marine economic fish with high food and ecological value, and its growth process has obvious male and female sexual dimorphism, with males growing significantly faster than females. However, the current sex identification technology is not yet mature, which will limit the growth rate of O. punctatus aquaculture and the efficiency of separate sex breeding, so the development of efficient sex molecular markers is imperative. This study identified a 926 bp DNA insertion fragment in the cdkn1/srsf3 intergenic region of O. punctatus males through whole-genome scanning, comparative genomics, and structural variant analysis. A pair of primers was designed based on the insertion information of the Y chromosome intergenic region in male individuals. Agarose gel electrophoresis revealed the amplification of two DNA fragments, 1118 bp and 192 bp, in male O. punctatus individuals. The 926 bp fragment was identified as the insertion in the intergenic region of cdkn1/srsf3 in males, while only a single 192 bp DNA fragment was amplified in females. The biological sex of the individuals identified in this manner was consistent with their known phenotypic sex. In this study, we developed a method to detect DNA insertion variants in the intergenic region of O. punctatus. Additionally, we introduced a new DNA marker for the rapid identification of the sex of O. punctatus, which enhances detection efficiency. The text has important reference significance and application value in sex identification, all-male breeding, and lineage selection. It provides new insights into the regulation of variation in the intergenic region of cdkn1/srsf3 genes and the study of RNA shearing.