Here, we present an immobilized enzyme cascade in a basket-type reactor allowing a one-pot, two-step enzymatic synthesis of L-erythrulose from D-serine and glycolaldehyde. Three enzymes, D-amino acid oxidase from Rhodotorula gracilis (DAAORg), catalase from bovine liver (CAT), and transketolase from Geobacillus stearothermophilus (TKgst) were covalently immobilized on silica monolithic pellets, characterized by an open structure of interconnected macropores and a specific surface area of up to 300 m2/g. Three strategies were considered: (i) separate immobilization of enzymes on silica supports ([DAAO][CAT][TK]), (ii) co-immobilization of two of the three enzymes followed by the third ([DAAO+CAT][TK]), and (iii) co-immobilization of all three enzymes ([DAAO+CAT+TK]). The highest L-erythrulose concentrations were observed for the co-immobilization protocols (ii) and (iii) (30.7 mM and 29.1 mM, respectively). The reusability study showed that the best combination was [DAAO + CAT][TK], which led to the same level of L-erythrulose formation after two reuse cycles. The described process paves the way for the effective synthesis of a wide range of α-hydroxyketones from D-serine and suitable aldehydes.