Abstract The effect of temperature on enantiomeric ratio (E) and specific reaction rate (rflow) in the continuous-flow mode acetylation of (±)-1-phenylethanamine (rac-1a), (±)-4-phenylbutan-2-amine (rac-1b) and (±)-1,2,3,4-tetrahydro-1-naphthalenamine (rac-1c) by variously immobilized Candida antarctica lipase B biocatalysts was studied in the 0–70 °C range. In the continuous-flow kinetic resolutions with differently immobilized CaLB biocatalysts, the character of temperature effect depended significantly both on the substrate and on the mode of immobilization. Alteration of E in the kinetic resolutions of three differently flexible amines rac-1a–c as a function of temperature was rationalized by the various flexibility of the lipase in its different forms. Our results indicated that the optimal method of immobilization depended both on the nature of the substrate and the reaction conditions.