A novel optical method is proposed for the determination of polymer glass transition temperature (Tg) by which the refractive index of polymer microspheres varying with temperature is monitored with the technique of microsphere imaging.It was demonstrated that the present method can eliminate most thermal lag and has sensitivity at least six fold higher than the conventional method in Tg determination.So the determined Tg is more accurate and varies less with heating rate than that obtained by conventional methods.The most attractive character of the new method is that it can simultaneously determine the Tg of several polymers in an experiment, thus greatly saving experimental time and heating energy.The method is not only applicable for polymer microspheres, but also for the materials with arbitrary shapes, therefore, it is expected to be broadly applied to different fundamental researches and practical applications of polymers.