The current study was conducted to evaluate the effect of different levels of single cell protein (SCP) and probiotic on performance, egg quality and immunological responses of laying hens. A total of 180 hen layers were used in a 3×3 factorial design allotted to one of 9 treatments, 4 replicates and 5 birds in cage. Dietary treatment consisted of three levels of SCP substitution with soybean meal (0, 30 and 60 %) and three levels of probiotic (0, 50 and 100 mg/kg of diet) that fed during at 80 d feeding trial includ 10 days for adaptation period and 70 days as main recording period. Performance parameters including feed intake, feed conversion ratio, egg production percentage, egg weight, egg mass and also egg quality were calculated on d 35 and 70 of experiment. To investigate immunological response, sheep red blood cells (SRBC) were injected twice (at 10 d intervals. After that, hens were killed at day 6 and 12 after the first and second injection, respectively. Moreover, Newcastle disease virus was injected and then sera were taken at day 6 after injection. In addition, differential leukocyte counts were evaluate at the end of experiment. On 70 day of experiment, two birds of each cage were exsanguinated to evaluate the relative weight of internal organs, enumeration of intestinal microbial counts and jejunal morphology. The results showed that using at least 30 % of SCP substitution for soybean meal caused remarkable decrease in serum cholesterol concentration and ileal pH (p 0.0001) as well as significant increase in antibody titer against Newcastle disease virus (p 0.01) and eggshell weight and strength (p 0.05). However, feeding 60% of SCP substitution led to decrease in serum triglyceride concentration (p 0.01), the ileal Escherichia coli enumeration (p 0.01), villus height to crypt depth ratio (p 0.0001) and egg mass (p 0.01). On the other hand administration of 50 mg/kg of probiotic caused remarkable decrease in serum cholesterol content (p 0.0001), SGPT (p 0.01) and ALP (p=0.08) enzyme activities, ileal Escherichia coli count (p 0.01), heterophile to lymphocyte ratio (p 0.0001), and also increase in villus height to crypt depth ratio (p 0.0001) and goblet cell numbers (p=0.016). Therefore our finding indicated that although supplementation of different levels of probiotic and 30 % of SCP substitution for soybean meal had no effects on productive performance, administration of the second levels of experimental factors led to improve in eggshell quality and immunological as well as intestinal histological responses in laying hens. Keywords : Single Cell Protein, Probiotic, Performance, Immunlogical responses, Intestinal histology, Laying hens