A fruit crop of significant economic importance, papayas are often propagated from seeds. Based on their freezing injury even at low moisture content, papaya seeds are classified as "intermediate seeds"; and rapid loss of viability in storage is assumed to be the primary cause of poor germination; thus, the development of appropriate technology is necessary to prolong the storability of papaya seeds. One such effective technology for prolonged seed storability is cryo-storage. The purpose of the current study was to investigate how cryostorage affects papaya seed storability. For this investigation, TNAU Papaya variety CO 8 seeds were utilised. Five distinct treatments were applied to the seeds, which were then kept for approximately six months at liquid nitrogen's vapour phase (-140°C). The biochemical parameters such as protein content, free amino acid, total carbohydrates, free sugars, total lipids, and dehydrogenase activity were measured on a monthly basis along with seed germination and seedling vigour in the cryopreserved papaya seeds. Results indicated that papaya seeds that had been vitrified (seeds with a 10% moisture content that had been soaked in loading solution for 20 minutes, then immersed in Plant Vitrification Solution 2 (PVS2) for another 20 minutes, packed in cryovials and kept in cryo-storage at -140°C) compared to papaya seeds that were packed in cloth bags and stored in ambient conditions had higher seed germination, dry matter production and vigour index at six months after storage. In comparison to fresh seeds, the results also showed that, after six months of cryo-storage, biochemical proximate, protein content, total carbohydrate content, total lipid content, and dehydrogenase activity were maintained in the aforementioned treatment with minimum reduction, while electrical conductivity, free amino acid content, and free sugar content gradually increased with the storage period at minimum rate. In crux, vitrified papaya seeds packaged in cryovials and kept in cryo-storage at -140°C preserved their quality for six months, meeting Indian Minimum Seed Certification Standards. This could be useful for storing papaya seeds for longer periods of time.