Polylactic acid (PLA) is a biodegradable polymer derived from renewable resources, showing potentials in replacing traditional petroleum-based polymers, yet its brittleness and low thermal-resistance limits its applications. Thus, glass fibers (GF) combined with heat treatment were used to prepare high-performance PLA. Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) were employed to analyze crystallization behavior of PLA/GF composite. Tensile, flexural and impact tests were conducted to investigate mechanical properties, and heat deflection temperature was measured to evaluate thermal resistance. GF can coincidently enhance strength, rigidity, and toughness of PLA. Isothermal heat treatment can further improve the mechanical properties regardless of GF content. Compared with neat PLA, the tensile strength, flexural modulus, and impact strength can be increased by 162.5%, 266.4%, 232.5%, respectively, in the presence of 20 wt% GF after isothermal heat treatment, and meanwhile heat deflection temperature can be increased from 50.6 °C to 148.8 °C. Both DSC and XRD analysis results indicated that GF can significantly enhance crystallization of PLA. Thus, not only GF but also enhanced crystallization led to the outstanding mechanical performance of PLA/GF composites. While GF shows little effect on thermal resistance, heat treatment can remarkably improve thermal stability, in particular for PLA/GF composites.