Background: Although there is an increasing need for eco-friendly and non-toxic food colorants, plant-based colors have shown to be a promising alternative to synthetic food colors. The natural pigment (betalain) was extracted from red beetroot utilizing a magnetic stirrer shaking apparatus in the current study. Objective: The purpose of this study was to evaluate the effect of betalain (natural pigment) and red dye 40 (synthetic pigment), on the chemical, microbiological, and sensory properties of ice cream. Materials and Methods: Betalain and red dye 40 were added into ice cream at variable concentrations of 50 mg/mL, 100 mg/mL, and 200 mg/mL. Specimens were stored for 70 days and evaluated every ten days. Results: The values of titratable acidity, PH, fat, protein, total count of microorganisms, and sensory characteristics were gave higher scores samples  than than in ice cream with red dye 40. Nevertheless, betalain doses of 50, 100, and 200 mg/mL have been reported to have a significant 70-day storage activity in ice cream. When compared to mixtures containing 50, 100, and 200 mg/mL of red dye 40 after 70 days. According to the findings, betalain may be utilized as a natural pigment and food preservative to boost stability during storage.Keywords: Red beetroot,Betalain, Functional foods,  Red Dye 40 (E129),Antioxidants, synthetic colors, Storage stability