Microalgae are photoautotrophic microorganisms known as producers of a large variety of metabolites. The taxonomic diversity of these microorganisms has been poorly explored. In this study, a newly isolated strain was identified based on the 18S rRNA encoding gene. The phylogenetic analysis showed that the isolated strain was affiliated with the Rhodomonas genus. This genus has greatly attracted scientific attention according to its capacity to produce a large variety of metabolites, including phycoerythrin. Growth and phycoerythrin production conditions were optimized using a Plackett–Burman design and response surface methodology. An expression profile analysis of the cpeB gene, encoding the beta subunit of phycoerythrin, was performed by qRT-PCR under standard and optimized culture conditions. The optimization process showed that maximum cell abundance was achieved under the following conditions: CaCl2 = 2.1328 g/L, metal solution = 1 mL/L, pH = 7 and light intensity = 145 μmol photons/m2/s, whereas maximum phycoerythrin production level occurred when CaCl2 = 1.8467 g/L, metal solution = 1 mL/L, pH = 7 and light intensity = 157 μmol/m2/s. In agreement, positive transcriptional regulation of the cpeB gene was demonstrated using qRT-PCR. This study showed the successful optimization of abiotic conditions for highest growth and phycoerythrin production, making Rhodomonas sp. suitable for several biotechnological applications.