Transketolase (TK) from S. cerevisiae was successfully immobilized on layered double hydroxides (LDH) using simple, affordable and efficient adsorption and coprecipitation based immobilization procedures. Optimization of the preparation was performed using zinc aluminium nitrate (Zn2Al-NO3) and magnesium aluminium nitrate (Mg2Al-NO3) LDH as immobilization supports, and the protein-to-LDH weight ratio (Q) was varied. The highest immobilization yields (98-99%) and highest relative specific activities (4.2-4.4 Umg−1 for the immobilized enzyme compared to 4.5 Umg−1 for the free enzyme) were both achieved when using a protein-to-LDH weight ratio (Q) of 0.38. Efficient lyophilization of the LDH-TK bionanocomposites thus synthesized was proven to allow easy use and storage of the supported TK with no significant loss of activity over a three-month period. The kinetic parameters of the LDH-TK enzyme were comparable to those of the free TK. The LDH-TK enzyme was finally tested for the synthesis of L-erythrulose starting from hydroxypyruvate lithium salt (Li-HPA) and glycolaldehyde (GA) as substrates. L-erythrulose was characterized and obtained with an isolated yield of 56% similar to that obtained with free TK. The reusability of the LDH-TK biohybrid material was then investigated, and we found no loss of enzymatic activity over six cycles