For the biological production of L-ribulose, conversion by enzymes or resting cells has been investigated. However, expensive or concentrated substrates, an additional purification step to remove borate and the requirement for cell cultivation and harvest steps before utilization of resting cells make the production process complex and unfavorable. Microbial fermentation may help overcome these limitations. In this study, we constructed a genetically engineered Candida tropicalis strain to produce L-ribulose by fermentation with a glucose/L-arabinose mixture. For the uptake of L-arabinose as a substrate and conversion of L-arabinose to L-ribulose, two heterologous genes coding for L-arabinose transporter and L-arabinose isomerase, were constitutively expressed in C. tropicalis under the GAPDH promoter. The Arabidopsis thaliana-originated L-arabinose transporter gene (STP2)-expressing strain exhibited a high L-arabinose uptake rate of 0.103 g/g cell/h and the expression of L-arabinose isomerase from Lactobacillus sakei 23 K showed 30% of conversion (9 g/L) from 30 g/L of L-arabinose. This genetically engineered strain can be used for L-ribulose production by fermentation using mixed sugars of glucose and L-arabinose.