Mimicking the green fluorescent protein (GFP), multicolor fluorescent polymers possessing enhanced fluorescence have been developed and applied to single-excitation cell imaging. The GFP core chromophore was covalently linked to the azide-functionalized amphiphilic block polymer poly(ethylene glycol) azide poly(methyl methacrylate). Through macromolecular assembly into micelles, the fluorescence enhanced and further increased with the elongation of poly(methyl methacrylate) chain due to the segmentation effect of the polymeric framework, which could reduce strong pi-pi interaction and suppress the chromophore's conformational motion. By a combination of chemically tailoring the core chromophore and macromolecular assembly strategy, multicolor fluorescent polymers showing a color palette from blue to orange were achieved under similar excitation conditions with the highest emission quantum yield approaching 8%, which is more than 80-fold larger than that of the core chromophore. Moreover, fluorescent emission color could be regulated by tuning the coassembling constitution of green and orange fluorescent polymers, generating three new types of emission color. Owing to their low cytotoxicity and good photostability, GFP-mimicking fluorescent polymers were suitable for single-excitation multicolor cell imaging, exhibiting maximum Stokes shift of 202 nm, ascribing to the effect of excited-state proton transfer (ESPT). More importantly, green, yellow, and orange fluorescent cell images were obtained from one single visual field, demonstrating identical information on examined cells, which would improve the accuracy and reliability of biological analysis.