Poly(D,L-lactic-co-glycolic acid) (PLGA) is one of the most commonly used drug carriers in nanomedicines because of its biodegradability, biocompatibility and low toxicity. However, the physico-chemical characterization and study of drug release are often lacking the investigation of the glass transition temperature (Tg), which is an excellent indicator of drug release behavior. In addition, the residual surfactant used during the synthesis of nanoparticles will change the glass transition temperature. We thus prepared PLGA nanoparticles with polymeric (poly(vinyl alcohol) (PVA)) and ionic (didodecyldimethylammonium bromide (DMAB)) surfactant to investigate their influence on the glass transition temperature. Determination of Tg in dry and wet conditions were carried out. The use of concentrated surfactant during synthesis resulted in a larger amount of residual surfactant in the resulting particles. Increasing residual PVA content resulted in an increase in particle Tg for all but the most concentrated PVA concentrations, while increasing residual DMAB content resulted in no significant change in particle Tg. With the presence of residual surfactant, the Tg of particle and bulk samples measured in wet conditions is much lower than that in dry conditions, except for bulk PLGA containing the ionic surfactant, which may be related to the plasticizing effect of the DMAB molecules. Notably, the Tg of both particles in wet conditions is approaching physiological temperatures where subtle changes in Tg could have dramatic effects on drug release properties. In conclusion, the selection of surfactant and the remaining amount of surfactant are crucial parameters to utilize in designing the physico-chemical properties of PLGA particles.