Stenocereus thurberi is a columnar cactus distributed in northwestern Mexico. Its study has focused on the phenology of the cactus and not on its fruit, called pitaya. The pitaya contains pigments, such as betalains, which are important bioactive compounds for their antioxidant capacity. Therefore, the extraction of betalains from pitaya pulp is an important step in providing information for the subsequent study and development of functional foods, nutraceuticals, colorants, and cosmetics. The objective of this study was to optimize the betalain extraction process from pitaya pulp (Stenocereus thurberi) in water and to evaluate its antioxidant capacity before and after its optimization using response surface methodology. In this study, a three-factor, three-level composite central design (CCD) with four center points was used to assess the influence of temperature, solvent volume, and sonication time on betalain extraction from pitaya pulp. Optimal extraction conditions were found using 57 °C, 34 mL of solvent, and 27 min of sonication. The total betalain content under optimal conditions was 2.6 mg/g of dry sample. The combination of the three factors showed high antioxidant activity by DPPH and ABTS. The conditions optimized for the extraction of betalains from the pitaya pulp in water by ultrasound did not affect its antioxidant capacity. Therefore, these conditions can be efficiently applied to extract betalains from pitaya pulp on a large scale as a promising alternative to replace synthetic dyes.