Articular cartilage focal lesion remains an intractable challenge in sports medicine, and autologous chondrocytes' implantation (ACI) is one of the most commonly utilized treatment modality for this ailment. However, the current ACI technique requires two surgical steps which increases patients' morbidity and incurs additional medical costs. In the present study, we developed a one-step cryopreserved off-the-shelf ACI tissue-engineered (TE) cartilage by seeding pellets of spheroidal cartilage stem/progenitor cells (CSPCs) on a silk scaffold. The pellets were developed through a hanging-drop method, and the incubation time of 1 day could efficiently produce spheroidal pellets without any adverse influence on the cell activity. The pellet size was also optimized. Under chondrogenic induction, pellets consisting of 40 000 CSPCs were found to exhibit the most abundant cartilage matrix deposition and the highest mRNA expression levels of SOX9, aggrecan, and COL2A1, as compared with pellets consisting of 10 000, 100 000, or 200 000 CSPCs. Scaffolds seeded with CSPCs pellets containing 40 000 cells could be preserved in liquid nitrogen with the viability, migration, and chondrogenic ability remaining unaffected for as long as 3 months. When implanted in a rat trochlear cartilage defect model for 3 months, the ready-to-use, cryopreserved TE cartilage yielded fully cartilage reconstruction, which was comparable with the uncryopreserved control. Hence, our study provided preliminary data that our off-the-shell TE cartilage with optimally sized CSPCs pellets seeded within silk scaffolds exhibited strong cartilage repair capacity, which provided a convenient and promising one-step surgical approach to ACI.