Rice is one of the most important staple food in the world. In many countries, environments with high temperature/humidity may provide favorable conditions for fungal propagation and production of aflatoxins during rice storage. Aflatoxin B1 (AFB1), one of the secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus, is particularly carcinogenic to humans. People might very likely cause severe illness once rice is contaminated by aflatoxins. Therefore, the objective of this study is to develop a nondestructive method for rapid and accurate detection of the AFB1 content in rice. In the present study, evaluation of AFB1 contaminated rice were conducted by using near infrared spectroscopy (NIRS) technique with chemometrics. NIR Spectra and the AFB1 contents of the artificially contaminated rice samples were measured by FOSS NIRS 6500 spectrometer and LC-MS/MS, respectively. Modified partial least square regression (MPLSR), a commonly used multivariate analysis method was then used to build calibration models. In summary, NIRS associated with chemometrics could provide a useful tool for rapid and accurate detection of AFB1 content in rice.