The chemoenzymatic synthesis of block copolymers from a bifunctional initiator using enzymatic ring-opening polymerization (eROP) and ATRP in two consecutive steps was investigated. First, a polycaprolactone (PCL) macroinitiator was obtained via an enzymatic ring-opening polymerization initiated by the bifunctional initiator. By carefully managing the water activity in the system, the amount of PCL not initiated by the bifunctional initiator was reduced to < 5%. Moreover, comparison of the results from H-1 NMR and MALDI-ToF of PCL obtained from different bifunctional initiators revealed an influence of the initiator structure on the initiation behavior in the enzymatic reaction. Block copolymers were obtained in a subsequent ATRP. The combination of various characterization techniques such as GPC, GPEC, and DSC provided clear evidence of the block structure of the polymers.