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.