The digestion and utilization of roughage by sheep depends on rumen digestion.The aim of this study was to evaluate the microbe quantity and fermentative efficiency of rumen supplement with molasses-urea. This experiment was conducted in 2014, eight sheep were selected and divided into two groups (a control group and a treatment group), and only the treatment group animals were supplied with molasses-urea for ad libitum consumption. Rumen fluid was collected every 2 h and rumen fermentation parameters were measured. The populations of majority bacteria were investigated by real-time PCR.The results showed that the populations of majority bacteria increased in the rumens of treatment group animals (P<0.05). Each bacterium quantity decreased gradually after feeding, and reached the lowest level 2 h after intake. It then slowly increased and reached the highest level at 8 h after intake. Finally, each bacterium quantity returned to the same level as before intake. In contrast, the protozoa number raised to the highest at 4 h after intake and declined gradually. The concentration of protozoa in the treatment group sheep was significantly higher than that of control group (P<0.05). The pH of rumen liquids was found in a normal range and was not different between both groups. However, the pH decreased from the highest level before feeding to the lowest level within 4 h, and it increased after intake for 8 h. The concentration of NH3-N and microbial crude protein (MCP) synthesis, in the rumen liquids, were both significantly higher than that of control group (P<0.05), the highest concentration of NH3-N and MCP was reached after feeding by 2 h and 4 h, respectively. Molasses-urea has a positive effect on the rumen, due to their favorable effect on rumen fermentation by the microbes in ruminant.