In order to illustrate their tolerance mechanism, four leading cultivars of Brassica napus (Mianyou 15, Guohaoyou 8, Yiyou 21, and Mianyou 16) were investigated to study the effects of Clopyralid, Benazolin, and Clethodium on physio-chemical and ultrastructural attributes of Brassica napus at during seed germination stage, by various concentrations (0.0, 0.5, 0.7, 1.0, 1.3 μg/10 mL) of Clopyralid, Benazolin, and Clethodium to rape seedlings in terms of various physiological and biochemical measurements and microscopic analysis. The main results were as follows: low concentration (0.5 μg/10 ml) of Clopyralid, Benazolin, and Clethodium had no significant effect on Brassica napus CVS. It was found that with the increasing of treated concentrations, the growth of Brassica napus was inhibited more when the concentration of Clopyralid, Benazolin, and Clethodium was higher than 1.3 μg·L^-1. It inhibited the net photosynthetic rate, chlorophyll content, chlorophyll fluorescence value, and anti-enzyme activities of Brassica napus. Moreover, it increased lipid peroxidation, accumulated reactive oxygen species, and damaged the ultrastructure of chloroplasts and mitochondria in mesophyll cells. In addition, the ability of resistance to the stress of Clopyralid, Benazolin, and Clethodium in all cultivars shows higher sensitivity which is more effective. Therefore, this study defined the effect of selected herbicides on Brassica napus, and the findings have great significance in extending the calculation by field application of those herbicides.