Objective This study was aimed to determine the effect of compound Chinese medicine ultrafine powders on egg laying performance, reproductive hormone, and related gene expression in laying hens.Method A total of 216 Xinyang black-feather laying hens (307-day-old) were selected and randomly divided into three groups with eight replicates in each group and nine in each replicate. The control group was fed the basal diet, and the experimental groups were fed basal diets supplemented with 0.5% Salvia miltiorrhiza (SM) + 0.25% Ligustrum lucidum (LL) + 0.25% Taraxacum mongolicum (TM)(compound 1), and 0.3% Leonurus japonicus (LJ) + 0.2% SM + 0.25% LL + 0.25% TM (compound 2) ultrafine powders, respectively. The pre-trial period was seven days, and the formal trial period was 120 days. Egg production performance, egg quality, and plasma reproductive hormone level were determined. The expressions of genes related to egg quality were detected by RT-PCR technology.Result Compared with the control group, during days 1-30, 31-60, and 1-120 of the trial, the egg laying rates of the compound 1 group increased by 7.56%, 4.00%, and 5.31% (P<0.05), respectively, and during 1-120 days the egg laying rate of the compound 2 group increased by 5.22% (P<0.05). On day 60 after treatment the plasma estradiol level and relative expression of oviduct PMCA1 mRNA increased by 21.45% and 77.00% (P<0.05), respectively, in the compound 1 group, and the relative expression of oviduct KCNA1 mRNA increased by 70.00% (P<0.05) in the compound 2 group. On day 90, the thickness of the eggshells increased by 12.12% and 9.09% (P<0.05), respectively, in the compound 1 and 2 groups. On day 120, the relative expressions of oviduct CA2 mRNA increased by 86.00% and 66.00% (P<0.05), respectively, in the compound 1 and 2 groups, and the relative expressions of oviduct CDH6, KCNA1 and SLC26A9 mRNA increased by 99.00%, 86.00% and 99.00% (P<0.05), respectively, in the compound 2 group.Conclusion Dietary supplementation with compound Chinese medicine ultrafine powders consisting of LJ, SM, LL, and TM could increase the egg production rate, improve egg quality of laying hen, increase the level of reproductive hormone, and up-regulate mRNA expressions of genes associated with eggshell formation. In addition, compound 1 presents the better effect.