This study explored the effects of different levels of hempseed meal(HSM) supplementation in laying hen diets, with and without a multienzyme supplement, on egg production, quality, yolk fatty acid composition. A total of 144 White Leghorn hens, aged 42 weeks, were divided into eight groups with six replicates each, and the experimental groups were fed with four HSM levels (0%,5%,10%,15%) with or without 1000mg multienzyme supplement per kg feed for a period of 84 days trial. Results indicated that with or without multienzyme did not significantly affect egg production, weight, mass, feed conversion ratio. Feed intake was consistent across HSM levels, but cubic interaction between multienzymes and HSM was observed(p<0.05). Eggshell parameters were unaffected by HSM or multienzyme supplementation. Yolk color(L* value) showed a cubic response to HSM levels, peaking in 5% treatment. The addition of multienzymes and HSM significantly decreased the a*value(p<0.05), while the b*value increased quadratically with HSM supplementation(p<0.05). Yolk fatty acid composition exhibited linear and cubic effects from HSM and multienzyme HSM interaction(p<0.05), with increased total polyunsaturated fatty acids(ƩPUFA) and decreased total monounsaturated fatty acids(ƩMUFA)(p<0.05). The omega-6/omega-3 ratio was linearly influenced by HSM supplementation(p<0.05). In summary, multienzyme supplementation alone did not significantly alter production or quality parameters, but interacted with HSM to affect feed intake, yolkcolor(a* value), and fatty acid composition. These findings suggest that HSM supplementation may improve the quality of egg yolk, although it does not affect overall egg production, and may therefore be viable option to improve the fatty acid composition of eggs.