β-carotene possesses significant biological activities, making it not only valuable as a nutritional supplement but also economically significant due to its coloring abilities. Today, β-carotene finds extensive applications across various industries, including pharmaceuticals, nutritional supplements, cosmetics, and animal feed additives. There are several approaches for producing β-carotene, encompassing natural extraction, chemical synthesis, and biosynthetic techniques. Among these, microbial fermentation stands out as particularly advantageous, given its natural and safer profile compared to traditional chemical synthesis methods. Microorganisms utilized for β-carotene production include native and genetically engineered strains of microalgae, bacteria, and fungi. Among native organisms, certain microalgae like Dunaliella have gained popularity due to their substantial β-carotene production capabilities. Additionally, specific bacterial species, notably Erwinia uredovora and Pantoea agglomerans, have been well recognized for their capacity to produce β-carotene. Furthermore, molds and yeasts have attracted considerable research attention, given their rapid growth rates and efficiency; particularly, Blakeslea trispora and Rhodotorula glutinis are recognized for industrial-scale β-carotene production. To further enhance productivity, significant advancements have been achieved through metabolic engineering, establishing microbial cell factories capable of high-yield β-carotene biosynthesis. The organisms predominantly employed as hosts in these engineered systems are Escherichia coli and Saccharomyces cerevisiae. This biotechnological approach provides notable benefits such as rapid production, shorter fermentation times, purity of the final product, tolerance to high β-carotene levels, and independence from spatial and environmental constraints. Moreover, this method has the potential to improve β-carotene bioavailability and facilitate its widespread commercial use. In this review, we comprehensively examine the biosynthetic production of β-carotene via natural microbial pathways, explore metabolic engineering strategies employed to enhance β-carotene biosynthesis, and discuss fermentation techniques and downstream processes designed to optimize production efficiency.