This study was conducted to evaluate the feeding value of the spent mushroom (Pleurotus eryngii) substrates (SMS) in laying hens (Hy-Line Brown). The fresh spent mushroom (Pleurotus eryngii) substrates collected from the DOJUN farm were fermented with Bacillus subtilis EJ3 for 2 weeks. A total of twenty-four laying hens were fed corn-soy based experimental diets containing 0% (control), 5% (T1), 10% (T2) and 15%(T3) fermented SMS for 7 weeks. There were no significant differences among the treatments in egg production, egg weight, egg mass, feed conversion and viability during the experimental period. Feed intake was significantly lowered in control (118.3 g) than T1 (121.9 g), T2 (120.3 g) and T3 (122.4 g). There were no significant differences among the treatments eggshell breaking strength, thickness and haugh unit, whereas the yolk color of T1, T2 and T3 were significantly heavy than T0. The palatability of boiled meat was significantly better in the T3 laying hens than in the T0 laying hens. In conclusion, fermented SMS can be used as resource of feed in laying hen feed at 5.0-15% level without effect on performance and egg qualify.