We report on the fabrication of multifunctional ratiometric probes for glucose and temperatures based on thermo-responsive poly(N-isopropylacrylamide) (PNIPAM) microgels covalently incorporated with glucose-recognizing moieties, N-acryloyl-3-aminophenylboronic acid (APBA), fluorescence resonance energy transfer (FRET) donor dyes, 4-(2-acryloyloxyethylamino)-7-nitro-2,1,3-benzoxadiazole (NBDAE), and rhodamine B-based FRET acceptors (RhBEA). P(NIPAM-APBA-NBDAE-RhBEA) microgels containing FRET pairs and APBA were synthesized via free radical emulsion copolymerization. The spatial proximity of FRET donors and acceptors within microgels can be tuned via thermo-induced microgel collapse or glucose-induced microgel swelling at appropriate pH and temperatures, leading to the facile modulation of FRET efficiencies. APBA moieties within P(NIPAM-APBA-NBDAE-RhBEA) microgels can bind with glucose at appropriate pH to form cyclic boronate moieties, which can decrease the pK(a) of APBA residues and increase the volume phase transition (VPT) temperature of microgels. The gradual addition of glucose into fluorescent microgel dispersions at intermediate temperatures, i.e., between microgel VPT temperatures in the absence and presence of glucose, respectively, can lead to the reswelling of initially collapsed microgels. Thus, P(NIPAM-APBA-NBDAE-RhBEA) microgels can serve as dual ratiometric fluorescent probes for glucose and temperatures by monitoring the changes in fluorescence emission intensity ratios. Moreover, P(NIPAM-APBA-NBDAE-RhBEA) microgels at pH 8 and 37 degrees C can serve as a ratiometric fluorescent glucose sensor with improved detection sensitivity as compared to that at 25 degrees C. MTT assays further revealed that thermoresponsive microgels are almost noncytotoxic up to a concentration of 1.6 g/L. These results augur well for the application of P(NIPAM-APBA-NBDAE-RhBEA) microgels for multifunctional purposes such as sensing, imaging, and triggered-release nanocarriers under in vivo conditions.