We report investigations of the response rates of bioresponsive hydrogel microlenses in order to gain a deeper understanding of their potential utility as a new label-free biosensing construct. Bioresponsive microgels were prepared from multi-responsive poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-AAc) microgels after conjugation with biotin (as a bound target) and aminobenzophenone (ABP) (as a photoaffinity label) via carbodiimide chemistry. Bioresponsive hydrogel microlenses were then formed from individual microgels via Coulombic assembly of the anionic microgels onto a positively charged glass substrate. Incubation with polyclonal anti-biotin resulted in antibody-cross-linked microlenses. The antibodies were then photoligated to the hydrogel network via UV irradiation, making the microlenses reversibly sensitive to free biocytin by disruption of biotin−antibiotin binding. The response rates of the microlenses to biocytin binding (bound antibody−antigen displacement) were studied by monitoring the microlens optical properties via brightfield optical microscopy. The response rates of the hydrogel microlenses are strongly coupled to analyte concentrations at an equilibrium number of antigen−antibody binding on the hydrogel microlenses. These results suggest that the hydrogel microlens construct may be a potential candidate for label-free biosensing/bioassay of protein and small molecules.