In order to explore the bio-interference of allelopathic rice with barnyardgrass under different nitrogen supplies, a new bioassay method, i.e., allelopathy-competition separation based (ACS) approach was recommended in this study. The results indicated that ACS could be successfully used to distinguish the rice allelopathy from its resources competition in rice-weed co-culture system, and to evaluate its response characteristics under different N supplies. The test rice accession PI312777 had a higher ability in interfering target weed barnyardgrass (BYD), represented by its stable and higher ability in resources competition, as well as its increasing allelopathic potential in suppressing companion weed BYD when the N supply decreased in the co-cultured system. The reverse was true in the case of rice accession Lemont, which had a lower allelopathic potential under all N supply levels, and a lower ability in resources competition under normal or rich N supply. But, Lemont had an obviously increased ability in resources competition under lower N supply, which was considered as the result of econiche competition in rice-weed system.