The present work investigated on carotenogenesis with high ${eta}$-carotene content by a new isolated high-activity strain-producer Rhodotorula acheniorum mutant MRN in cheese whey ultrafiltrate. After a serial of UV, ethymethanesurfonate (EMS), and nitrosoguanidine (NTG) mutagenesis, a mutant named MRN of the red lactosepositive yeast strain R. acheniorum was obtained. Then, the effects of different growth medium factors on carotenoid production by this mutant at batch-scale level were identified and optimized by means of response surface methodology (RSM) in order to achieve high-level production of ${eta}$-carotene. The optimum conditions required to achieve the highest level of ${eta}$-carotene ($262.12{pm}1.01$ mg/L) were determined as follows: whey ultrafiltrate (WU) lactose concentration 55 g/L, pH 5.85, ammonium sulfate concentration 3.5 g/L, temperature $23^{circ}C$, and aeration rate 1.56 vvm. The medium optimization resulted in a 6.45-fold increase in volumetric production ($262.12{pm}1.01$ mg/L) and a 4.62-fold increase in the cellular accumulation ($10.69{pm}0.19$ mg/g) of ${eta}$-carotene.