The selection of stably expressed reference genes is crucial for quantitative real-time polymerase chain reaction (qRT-PCR) assay when assessing osteosarcoma treated with cyclophosphamide (CTX) and Ginkgo biloba L. extract (EGB). We aimed to identify the reference genes that are stably expressed in human osteosarcoma cell lines after the above treatments. We used qRT-PCR assay to determine the expression levels of 18S, ACTB, ALAS1, GAPDH, TBP, HPRT1, RPL29, HMBS, PPIA, PUM1, GUSB, and B2M in the cell lines MG-63, Saos-2, and U2OS, which were treated with CTX or EGB or both. We analyzed the expression stability of these genes using geNorm, NormFinder, and BestKeeper. The optimal reference gene combinations were 18S + PPIA + HPRT1 in the total group, HMBS + ALAS1 + 18S in the EGB-treated group, PPIA + RPL29 in the EGB + CTX-treated group, PPIA + HPRT1 in the MG-63 group, 18S + PPIA in the Saos-2 group, and HPRT1 + ALAS1 + GAPDH in the U2OS group. We conclude that no single reference gene was most stably expressed in all three treated cell lines. Our findings provide a suitable approach through which qRT-PCR can be applied to investigate the pharmacological effects and the molecular mechanisms of CTX and EGB on human osteosarcoma.