Open biomass burning (BB) has contributed severely to the ambient levels of particulate matter of less than 2.5 μm diameter (PM2.5) in upper northern Thailand over the last decade. Some methods have been reported to identify the sources of burning using chemical compositions, i.e., ions, metals, polycyclic aromatic hydrocarbons, etc. However, recent advances in nuclear techniques have been limited in use due to their specific instrumentation. The aims of this study were to investigate the sources of ambient PM2.5 in Chiang Mai city using stable carbon (δ13C) and nitrogen isotopes (δ15N). The mean concentrations of total carbon (TC) and total nitrogen (TN) in PM2.5 were 12.2 ± 5.42 and 1.91 ± 1.07 μg/m3, respectively, whereas δ13C and δ15N PM2.5 were −26.1 ± 0.77‰ and 10.3 ± 2.86‰, respectively. This isotopic analysis confirmed that biomass burning was the source of PM2.5 and that C3 and C4 plants contributed about 74% and 26%, respectively. These study results confirm that the stable isotope is an important tool in identifying the sources of aerosols.