This study explores the synthesis, characterization, and application of a heterofunctional initiator derived from 2-hydroxypropyl cyclodextrin (HP-β-CD), having eight bromoester groups and thirteen hydroxyl groups allowing the synthesis of mikto-arm star-shaped polymers. The bromoesterification of HP-β-CD was achieved using α-bromoisobutyryl bromide as the acylation reagent, modifying the cyclodextrin (CD) molecule as confirmed by electrospray ionization mass spectrometry (ESI-MS), nuclear magnetic resonance (NMR), attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy analysis, and differential scanning calorimetry (DSC) thermograms. The initiator’s effectiveness was further demonstrated by obtaining star-comb and mikto-arm polymers via an enzymatically assisted atom transfer radical polymerization (ATRP) method and subsequent ring-opening polymerization (ROP). The ATR polymerization quality and control depended on the type of monomer and was optimized by the way of introducing the initiator into the reaction mixture. In the case of ATRP, high conversion rates for poly(ethylene oxide) methyl ether methacrylate (OEOMA), with molecular weights (Mn) of 500 g/mol and 300 g/mol, were achieved. The molecular weight distribution of the obtained polymers remained in the range of 1.23–1.75. The obtained star-comb polymers were characterized by different arm lengths. Unreacted hydroxyl groups in the core of exemplary star-comb polymers were utilized in the ROP of ε-caprolactone (CL) to obtain a hydrophilic mikto-arm polymer. Cloud point temperature (TCP) values of the synthesized polymers increased with arm length, indicating the polymers’ reduced hydrophobicity and enhanced solvation by water. Atomic force microscopy (AFM) analysis revealed the ability of the star-comb polymers to create fractals. The study elucidates advancements in the synthesis and utilization of hydrophilic sugar-based initiators for enzymatically assisted ATRP in an aqueous solution for obtaining complex star-comb polymers in a controlled manner.