The nucleation of flocculent sludge as seed in a mesophilic Internal Circulation Anaerobic reactor was investigated with a quantitative method proposed by us in this paper. In the 85th days, the average sludge diameter increased from 47.8 microm to 96.1 microm, which indicated that the nucleation was completed. During this process, the average sludge diameter had a significant linear correlation with the time with coefficient of 0.9893, the increase rate of average sludge diameter was 0.58 microm/d. Nucleus ratio of sludge increased from 7.6% on the 1st day to 36.1% on the 85th day. The increase rate of the nucleus ratio fluctuated in a wide range, experienced 'fast-slow-fast' three stages. During the experiment, the concentration of extra cellular polymers (ECPs) of the sludge was significantly positively correlated with the increase rate of the nucleus ratio, which indicated that ECPs could affect nucleation rate. During nucleation, the bio-activity of the sludge increased, but the improvement of settlement ability was not significant. The above-mentioned quantitative method and results would be useful to understand the sludge granulation mechanism on the level of reactor.