Poly (DL-lactic-co-glycolic acid) (PLGA) microparticles represent a promising formulation approach for providing steady pharmacokinetic/pharmacodynamic profiles of therapeutic drugs for a long period. Understanding and controlling the supramolecular structure of PLGA microparticles at a molecular level is a prerequisite for the rational design of well-controlled, reproducible sustained-release profiles. Herein, we reveal that the role of PLGA molecular conformation in particle formation and drug release. The nanoscale mobility of PLGA microparticles spray-dried using the solvents with distinct polarities was investigated by using nuclear magnetic resonance (NMR) and neutron scattering. By employing chemometric method, we further demonstrated the evolution of nanoscale networks in spray-dried PLGA microparticles upon the water absorption. Our results indicate that PLGA molecules form more chain entanglements during spray drying when using the solvents with low polarity, where PLGA molecule adopts a more flexible, extended conformation, resulting in that the more resistant network against the water absorption in the spray-dried PLGA microparticles. This work underlines the role of PLGA molecular conformation in controlling formation and evolution of nanoscale network of spray-dried PLGA microparticles and will have important consequences in achieving the customized drug release from PLGA microparticles.