Ceramides are spingolipid compounds that are very attractive as active components in both the phar-maceutical and the cosmetic industries. In this study, the synthesis of ceramide analogs, the so-calledpseudo-ceramides, was carried out using for the first time a two-step continuous enzymatic processwith immobilized Candida antarctica lipase B (Novozym®435) in a packed-bed bioreactor. The first stepinvolved the selective N-acylation of 3-amino-1,2-propanediol using stearic acid as the first acyl donor(i). This was followed by the selective O-acylation of the N-stearyl 3-amino-1,2-propanediol synthesizedin the first step, with myristic acid as the second acyl donor, to produce a N,O-diacyl 3-amino-1,2-propanediol-type pseudo-ceramide, namely 1-O-myristyl,3-N-stearyl 3-amino-1,2-propanediol (ii). Theprocess was first optimized by evaluating the influences of three factors: feed flow rate, quantity of bio-catalyst and substrate concentration. Under optimal conditions an amide synthesis yield of 92% and asatisfying production rate of almost 3.15 mmol h−1gbiocatalyst−1(1128 mg h−1gbiocatalyst−1) were obtained.The second step, N-acyl 3-amino-1,2-propanediol O-acylation, was similarly optimized and in additionthe effect of the substrate molar ratio was studied. Thus, an optimal pseudo-ceramide synthesis yield of54% and a production rate of 0.46 mmol h−1gbiocatalyst−1(261 mg h−1gbiocatalyst−1) were reached at a 1:3ratio of amide to fatty acid. In addition, it was demonstrated that this two-step process has great potentialfor the production of N,O-diacyl 3-amino-1,2-propanediol-type pseudo-ceramides on an industrial scale.It was shown in particular that Novozym®435 could be used for more than 3 weeks without a drop inthe yield during the first step of 3-amino-1,2-propanediol N-acylation, proving that this biocatalyst isvery stable under these operational conditions. This factor would greatly reduce the need for biocatalystreplacement and significantly lower the associated cost.