This paper describes a useful synthetic strategy based on sequential enzyme-catalyzed aldol addition reactions. The aldol reaction of an acceptor aldehyde with acetaldehyde, catalyzed by 2-deoxyribose-5-phosphate aldolase (DERA, EC 4.1.2.4), gives a new aldehyde, which subsequently reacts with a second donor substrate, catalyzed by DERA or another aldolase. This sequential aldol reaction proceeds because of the formation of a stable cyclic hemiacetal or hemiketal product. In this way several 6-substituted 2,4,6-trideoxyhexoses have been stereoselectively synthesized via a one-pot double DERA-catalyzed reaction. DERA coupled with fructose-1,6-diphosphate aldolase from rabbit muscle (RAMA, EC 4.1.2.13) in a one-pot reaction gave a variety of 5-deoxyheptuloses, however not in 100% stereoselectivity. The sequential aldol reactions with four substrates were accomplished by coupling DERA with N-acetylneuraminic acid aldolase (NeuAc aldolase, EC 4.1.3.3) to give several nine-carbon sialic acid-type sugar derivatives.