A study was conducted to evaluate enzyme and probiotic feed supplements as alternative feed additives to alleviate the problems associated with the withdrawal of antibiotics from layer diet. A total of 180 Hy-Line W-98 hens, 40 weeks old, were assigned into four treatment groups so that there were 45 laying hens in each group. Four dietary treatments were randomly assigned to the four groups of hens. Hens in Group 1 were fed basal diet only (negative control), Group 2 was fed basal diet ' antibiotic at a level of 2.0 g/kg (positive control), Group 3 was fed basal diet ' multi-enzyme at a level of 2.0 g/kg (ES) and Group 4 was fed basal diet ' multi-species probiotic at a level of 0.5 g/kg (PS). The results showed that addition of either enzyme or probiotic supplementation to the basal diet significantly increased the egg production, egg weight and egg mass of hens than the negative control group. There was no significant difference in feed intake among treatments, although the feed conversion improved with the addition of antibiotic, enzyme and probiotic than the negative control. Over the 70-day trial period, weight gain was higher than the negative control for birds fed the diet containing probiotic. There was no significant effect on mortality during the study. Haugh unit score was significantly increased in the groups fed the ES and PS diets when compared with negative control group. Egg shape index, specific gravity, yolk index, shell weight, shell thickness and damaged eggs did not differ significantly among the laying hens of all dietary groups. High-density lipoprotein-cholesterol was significantly increased by probiotic supplementation compared with other dietary groups. Dietary treatments did not influence eosinophils and monocytes, increased lymphocyte and decreased heterophil and H:L ratio. The geometric means haemagglutination inhibition titres of birds fed on diets containing antibiotic, enzyme and probiotic supplementation were higher from 28 to 70 days than that of negative control. It may be concluded that the supplementation of enzyme and probiotic in layer diets did not appear to cause any adverse effects on egg production, egg quality and immunity compared with the negative control laying hens and non-antibiotic feed additives can be used as alternative. Moreover, the dietary supplementation of probiotic may lead to the development of low-cholesterol chicken eggs as demanded by health-conscious consumers.