In this work, the effect of Cr doping on the mechanical properties of Mo2FeB2-based cermets was studied. The results indicated that the hardness was continuously enhanced with the Cr contents, while the transverse rupture strength increased and then decreased. With the increase of Cr contents, the decreased binder deteriorated the fracture toughness. In the Mo2FeB2 particles of the cermets, Cr atoms mainly substituted the large-size Mo atoms instead of Fe. However, the first-principles calculation results showed that when the Cr atoms occupied the sites of Fe, the Cr-doped Mo2FeB2 were more thermodynamically stable. Based on the elemental composition of Cr-doped Mo2FeB2 bulks, the sites of Cr in Mo2FeB2 were mainly determined by the contents of Mo and Fe. In the Mo-depleted Mo2FeB2 bulk, Cr atoms preferred to substitute the Mo atoms, while Cr atoms occupied the sites of Fe in the Fe-depleted Mo2FeB2 bulk. Hence, the relatively lower content of Mo in Mo2FeB2-based cermets resulted in the substitution of Mo with Cr atoms. The intrinsic hardening of Mo2FeB2 induced by Cr doping contributed to the enhanced hardness of Mo2FeB2-based cermets. Besides, the decreased bulk modulus and shear modulus were conducive to the improvement of the transverse rupture strength.