The prevalence of synthetic colorants in commercial products has raised concerns regarding potential risks, including allergic reactions and carcinogenesis, associated with their use or consumption. Natural plant extracts have gained attention as potential alternatives. This research focuses on callus induction and the establishment of cell suspension cultures from Celosia argentea var. plumosa. Friable callus was successfully induced using hypocotyl explants cultured on semi-solid Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1 mg/L 6-benzylaminopurine (BAP). The friable callus cell line was used to establish a suspension culture. The effects of sucrose, BAP, and tyrosine concentrations on betalain production were investigated using response surface methodology (RSM) based on central composite design (CCD). Optimal conditions (43.88 g/L sucrose, 0.15 mg/L tyrosine, and 0.77 mg/L BAP) yielded 43.87 mg/L total betalain content after 21 days, representing a threefold increase compared to the control. BAP had a significant positive impact on betalain production, and increasing BAP and sucrose concentrations generally led to higher betalain production. However, tyrosine was not a significant factor for betalain production in cell suspension cultures. Additionally, antioxidant assays showed that suspension-cultured cells (SCCs) under optimized conditions exhibited free radical scavenging activity comparable to that observed in C. argentea var. plumosa flower extract. This study indicates the potential for further research on betalain production using C. argentea var. plumosa cell cultures, which may have commercial applications.