Crop allelopathy provides a viable alternative in managing resistant weed populations. A successful demonstration of an allelopathic interaction is needed to be separated from resource competition. A reliable laboratory screening technique can test this chemical interaction between a donor and a receiver species. However, the standardization of any screening tactic for a new species is really challenging. In this study we optimized the simple root exudates bioassay, Equal Compartment Agar Method (ECAM), for canola through basic features including sowing patterns, density, and distance between canola and annual ryegrass. We established that all of the above features have major roles on canola seedling interference ability (allelopathy + competition). Due to the combined effect of allelopathy and competition, the zigzag canola sowing pattern enhanced the inhibitory effects of canola seedlings more than other sowing patterns, i.e. circular, parallel, and cone. The sowing distance of canola played a major role on root growth of annual ryegrass in that the nearer the receiver annual ryegrass plants to the donor canola seedlings, the greater were the toxic effects. Canola sowing distance at 1, 2, and 3 cm inhibited the root growth of ryegrass by 50, 46, and 36%, respectively. Results also showed that root growth of ryegrass decreased with increased canola densities (0 - 40 seedlings/beaker). Furthermore, the added carbon experiment showed that the inhibitory effects of canola were diluted by added carbon in agar growth medium, indicating canola root exudates acted directly on the root growth of annual ryegrass. This study suggests parameters such as canola sowing pattern, density, and sowing distance between canola and test weed species need to be considered for designing canola allelopathy bioassay through ECAM under laboratory conditions.