β-carotene is a commonly used food colorant. In this work, a novel β-carotene producing strain, Serratia marcescens RB3, was isolated and identified by physiological and biochemical tests, as well as 16S rDNA sequence analysis. The production of β-carotene by S. marcescens RB3 was identified through HPLC analysis. The cultivation conditions for β-carotene production by S. marcescens RB3 were optimized as 2.0% lactose, 2.0% peptone, 0.3% beef extract, 1.0% NaCl supplemented with 0.05% Fe²⁺, pH 6.0 and 30ºC. Under the optimal conditions, the yield of β-carotene achieved 2.45 µg/mL.