Lubricant plays an important role in machining. Due to the problems of environmental protection and workers' health, traditional lubricants are gradually replaced. Nanofluid, adding nanoparticles into base oil, is a kind of environment-friendly low-carbon lubricant, which improves not only the heat transfer capacity but also the lubrication performance. However, how many nanoparticles added into the fluid can improve the lubrication performance best? There are few studies on the mechanism of lubrication performance of different concentrations, and the results are not very clear. In this research, nanofluid as lubricating fluid is made by adding 0.5~2.0% mass concentration of Al 2 O 3 nanoparticles to the cottonseed oil during milling Grade 45 steel in minimum quantity lubrication (MQL) mode. The tribological properties of nanofluids with different concentrations at the tool/workpiece interface were studied by macro evaluation parameters (specific energy) and micro evaluation parameters (surface roughness, micro morphology, contact angle and viscosity). The results show that the concentration of nanoparticles has the most optimal value. When the concentration is 0.5 wt%, the specific energy is the minimum (0.281), and the roughness value is the lowest (1.63 μ m) . Meanwhile, slender chip and the largest droplet wetting area are obtained, which indicates better tribological properties. There is a nonlinear relationship between the lubricating effect of nanofluid and its concentration. The proper concentration of Al 2 O 3 nanofluid has good tribological properties and more potential to replace the pouring lubrication.