In order to further reduce particulate matter from diesel engine fuelled with biodiesel. Raman spectroscopy was used to investigate the microstructure of particulate matters with and without exhaust gas recirculation (EGR). Five-band fitting curves were adopted for first-order Raman spectrum. In addition, graphite crystallite size and C—C bond distance in graphite lattice were calculated. The results showed that D1 full width at half maximum (FWHM) increased and ID1/IG decreased with the increasing of blend of biodiesel. D1 FWHM increased and ID1/IG increased with EGR. These showed particulate matters from biodiesel and blend oil had higher chemical heterogeneity and more ordered graphite structure. EGR made particulate matters become higher chemical heterogeneity and less ordered graphite structure. The main kind of particles’ defect was located at the edge of graphite. The concentration of defect, volatile organic compounds (VOF) in particulate matters increased with biodiesel addition or EGR, indicating oxidation reactivity of particulate matter. Biodiesel addition to diesel made graphite crystallite size larger and EGR made graphite crystallite size smaller. In addition, biodiesel and EGR had slight influence on C—C bond distance in graphite lattice. The C—C bond distance in graphite lattice is about 0.142 nm.