Articular cartilage lacks the capacity for self-repair after injury, rendering autologous chondrocyte transplantation a crucial treatment option for severe cartilage damage. However, the in vitro expansion of chondrocytes in elderly patients poses challenges, particularly due to inadequate cell numbers and rapid phenotypic loss, often exacerbated by the use of animal-derived serum. In this study, we propose the utilization of human autologous serum (HAS) as a supplement in the in vitro cell culture to preserve the phenotype of chondrocytes isolated from the same patient. Chondrocytes cultured in medium supplemented with 5% HAS demonstrated improved cell proliferation and adherence, whereas a 10% concentration promoted a more differentiated phenotype. While 10% fetal bovine serum stimulates chondrocyte proliferation in the short term, 10% HAS is more advantageous for the long-term viability of cells. Notably, chondrocytes treated with the optimal 10% HAS supplementation exhibited a higher level of autophagy compared to those treated with 5% HAS alone. Our findings suggest that 10% human autologous serum holds promise for the efficient expansion of patient-derived autologous chondrocytes and facilitates the maintenance of chondrocyte phenotype, in part through enhanced cellular autophagy.