The instability of secondary metabolite production is a ubiquitous problem in plant cell culture. In order to understand the instability in plant cell culture, we investigated anthocyanin accumulation in suspension cultures of Vitis vinifera, as a model system, in our laboratory. Not only the anthocyanin contents but also its composition exhibited instability along with the long-term subculture. New methods were developed to indicate the instability of plant cell culture. Both the definition of instability coefficient (δ) and the application of factor scores were the first time in this field. To examine the effects of culture conditions on instability of anthocyanin biosynthesis, different subculture cycles and inoculum sizes had been investigated. Subculture cycle and inoculum size were both environmental cues driving the instability. Compared with subculture cycle, inoculum size was more effective in working on the instability of anthocyanin accumulation. Among all the conditions investigated in our study, (6.5 d, 2.00 g), (7 d, 2.00 g), (7.5 d, 2.00 g), (7 d, 1.60 g) and (7 d, 2.40 g), the condition of 7 d-subculture cycle together with 1.60 g-inoculum size was the best one to keep the stable production of anthocyanins.