Abstract: Separate pond rearing of males and females combined with scheduled artificial insemination (AI) serves as the foundation for improving utilization of elite broodstock and family-based breeding of Chinese soft-shelled turtle (Pelodiscus sinensis). Effective in vitro sperm preservation is a critical step enabling AI implementation. We systematically screened the conditions and extender components for sperm cooled storage. The results show that the optimal preservation temperature and pH were 0–4 °C and 7.0–7.4, respectively. Among the six common aquatic extenders screened, Hanks' solution was identified as the optimal basal extender. A composite extender formulated by supplementing Hanks' solution with 50% (φ) Percoll and 5% (φ) homologous serum provided a synergistic protective effect, further enhancing sperm preservation. After 21 d of storage, sperm motility remained above 40%. Furthermore, histological staining confirms that the optimized extender better preserved sperm structural integrity. In conclusion, Percoll and serum exhibit a synergistic protective effect, jointly maintaining sperm structural stability. This study successfully develops an extender suitable for in vitro preservation of P. sinensis sperm for cooled storage, providing practical technical support for artificial insemination and family-based breeding in this species.