In this work we explored an enzyme-mediated method for selective and efficient decoration of aqueous microgels with biomolecules. Poly(N-vinylcaprolactam) (VCL) microgels with varied amounts of glycidyl methacrylate (GMA) as comonomer incorporated in the microgel shell were synthesized and characterized in regard to their size, swelling degree, and temperature-responsiveness in aqueous solutions. The surface of the PVCL/GMA microgel containing 5 mol % glycidyl methyacrylate was modified by grafting of a specific recognition peptide sequence (LPETG) for Sortase A from Staphylococcus aureus (Sa-SrtAΔ59). Sortase-mediated conjugation of the enhanced Green Fluorescent Protein (eGFP) carrying a N-terminal triglycine tag to LPETG-modified microgels was successfully performed. Conjugation of eGFP to the microgel surface was qualitatively proven by confocal microscopy and by fluorescence intensity measurements. The developed protocol enables a precise control of the amount of eGFP grafted to the microgel surface as evidenced by the linear increase of fluorescence intensity of modified microgel samples. The kinetic of the sortase-mediated coupling reaction was determined by time-dependent fluorescence intensity measurements. In summary, sortase-mediated coupling reactions are a simple and powerful technique for targeted surface functionalization of stimuli-responsive microgels with biomolecules.