Most human serum albumin (HSA) for medical applications is derived from human plasma due to the lack of suitable heterologous expression systems for recombinant HSA (rHSA). To determine whether plant cell cultures could provide an alternative source, we employed the hyper-translatable cowpea mosaic virus protein expression system (CPMV-HT) to stably express rHSA in tobacco Bright Yellow-2 (BY-2) cells. rHSA was stably produced with yield up to 11.88 μg/ml in the culture medium, accounting for 0.7% of total soluble protein, in a 25-ml flask. Cultivation of transgenic cells in modified Murashige and Skoog medium with a pH of 8.0 improved the yield of rHSA two-fold, which may be the result of reduced proteolytic activity in the modified medium. A simple purification scheme was developed to purify the rHSA from culture medium, resulting in a recovery of 48.41% of the secreted rHSA. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and N-terminal sequence analysis of the purified rHSA revealed that plant cell-derived rHSA is identical to that of the plasma-derived HSA. Our results show that the CPMV-HT system, which was originally developed as a transient expression system for use in whole plants, can also be used for high-level expression of rHSA, a protein highly susceptible to proteolysis, in transgenic tobacco cells.