Smart polymers or reversibly soluble–insoluble polymers (cationic starch and Eudragit L-100) were coated onto the wells of a 96-well microplate. These polymers could selectively (i) bind proteins (cationic starch bound to maltose binding protein [MBP] and Eudragit L-100 bound to lactate dehydrogenase [LDH]) from their crude solution and (ii) refold proteins from their solubilized inclusion bodies. The purified/refolded proteins bound to the wells showed fluorescence emission spectra identical to their solution spectra. The purified wild-type MBP showed a single band on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The refolded MBP mutant (MBP224D) was found to be active as determined by fluorescence-based maltose binding assay. MBP (with an equilibrium constant [Kd] of 1.4μM for maltose binding) could sense maltose concentration in the range of 0.1 to 2.0μM. Similarly, lactate dehydrogenase (with an equilibrium constant of 6.1×10−4M for lactate binding) could sense lactate in the concentration range of 10 to 200μM. The main thrust of the work is to integrate protein expression, purification and refolding (if necessary), immobilization, and analyte sensing in a 96-well plate format.