l-Ribose, a starting material for the synthesis of l-nucleoside, has attracted lots of attention since l-nucleoside is responsible for the antiviral activities of the racemic mixtures of nucleoside enantiomers. In this study, the l-ribulose-producing Candida tropicalis strain was engineered for the conversion of l-arabinose to l-ribose. For the construction of a uracil auxotroph, the URA3 gene was excised by homologous recombination. The expression cassette of codon-optimized l-ribose isomerase gene from Acinetobacter calcoaceticus DL-28 under the control of the GAPDH promoter was integrated to the uracil auxotroph. The resulting strain, K1 CoSTP2 LsaAraA AcLRI, was cultivated with the glucose/l-arabinose mixture. At 45.5 h of fermentation, 6.0 g/L of l-ribose and 3.2 g/L of l-ribulose were produced from 30 g/L of l-arabinose. The proportion between l-ribose and l-ribulose was approximately 2:1 and the conversion yield of l-arabinose to l-ribose was about 20% (w/w). The l-ribose-producing yeast strain was successfully constructed for the first time and could convert l-arabinose to l-ribose in one-pot fermentation using the mixture of glucose and l-arabinose.