Microalgae are a potential source of a wide range of food and novel value‐added products. The versatility of microalgae to produce different kind of pigments is gaining interest as a sustainable source of natural carotenoids. Currently, commercial production of carotenoids from selected microalgae requires special culture conditions which are difficult to maintain. The present study has been undertaken to optimize culture conditions for growth and carotenoid production by a new isolate Scenedesmus quadricauda PUMCC 4.1.40. The results revealed that test organism produced 1.54 mg dry biomass/ml with a content of 40 μg carotenoids/mg dry biomass during stationary phase. The growth and carotenoid production was increased by 2.4‐fold under combined optimized culture conditions. The optimized conditions were growth medium, Chu‐10; pH 8.5; temperature, 30°C; nitrogen, 20 mM nitrate; phosphate, 0.22 mM; NaCl, 0.42 mM and blue light. Separation and identification of four important carotenoids through high‐performance thin‐layer chromatography (HPTLC) followed by purification using flash chromatography and quantification by HPLC revealed 23.8, 19.0, 6.5, and 4.0 μg astaxanthin, β‐carotene, lutein, and canthaxanthin /mg dry biomass, respectively. The amount of total carotenoids (98 μg/mg dry biomass) containing 40% valuable astaxanthin and β‐carotene produced under optimized conditions was significantly higher than control cultures. This justifies that S. quadricauda is a promising candidate for scale‐up production of carotenoid.