Bio-nanocomposites based on poly (lactic acid)/epoxidized natural rubber (PLA/ENR) blends containing graphene (G) nanofiller were prepared by melt compounding using nano-single screw extruder with a screw speed of 60 rpm, and followed by compression molding. The ENR and graphene loading was varied from 0-30 wt% and 0.5-2.0 phr. The objectives of this work are to investigate the effect of ENR and graphene loading on PLA matrix on the thermal, morphology and toughness properties. The TGA curve of PLA/ENR and PLA/ENR/G bio-nanocomposites shows a single step of thermal degradation. As increasing the loading of graphene, degradation temperature (Tdeg) was increase where graphene act to slow down the rate of degradation. The PLA/ENR blends showed a co-continuous morphology while PLA/ENR/G bio-nanocomposites revealed a good dispersion of graphene in a PLA/ENR matrix at 1 phr of graphene loading. The optimum impact strength was recorded at 10 wt% of the ENR loading which is 104.6 J/m for the PLA/ENR system. In the case of PLA/ENR/G system, the optimum impact strength was recorded at 1 phr of graphene which is 219.5 J/m. The thermal and toughness enhancement of PLA/ENR/G bio-nanocomposites will diversify the future application of this bio-nanocomposites.