Rare sugar is a kind of important low-energy monosaccharide that is rarely found in nature and difficult to synthesize chemically. D-allose, a six-carbon aldose, is an important rare sugar with unique physiological functions. It is radical scavenging active and can inhibit cancer cell proliferation. To obtain D-allose, the microorganisms deriving D-psicose 3-epimerase (DPE) and L-rhamnose isomerase (L-RhI) have drawn intense attention. In this paper, DPE from Clostridium cellulolyticum H10 was cloned and expressed in Bacillus subtilis, and L-RhI from Bacillus subtilis 168 was cloned and expressed in Escherichia coli BL21 (DE3). The obtained crude DPE and L-RhI were then purified through a HisTrap HP affinity chromatography column and an anion-exchange chromatography column. The purified DPE and L-RhI were employed for the production of rare sugars at last, in which DPE catalyzed D-fructose into D-psicose while L-RhI converted D-psicose into D-allose. The conversion of D-fructose into D-psicose by DPE was 27.34%, and the conversion of D-psicose into D-allose was 34.64%.