Annually, the large volumes of paper mill effluent to the environment in Iran. The major pulp and paper mill contain a large amount of alcohols and fatty acids and resin acids. Such materials stimulate vitellogenin production and suggested to be an estrogen mimic which cause sterility, sex reversal, yolk advance of replication and disruption of hormone cycles in fishes. Recently, vitellogenin gene quantification is considered as an efficient biomarker for the detection of estrogenic wastewater. Therefore, in this study the real time quantification of vitellogenin gene was used to evaluate the impact of pulp and paper mill on rainbow trout. After LC₅₀ detection, the fish was exposed to 200 and 500 ppm (0.1 and 0.25 LC₅₀, respectively) of the pulp and paper mill obtained from Mazandaran and Pars ahvaz company- Iran for 6 and 14 days and compared with control. To evaluate vitellogenin gene expression, the liver was taken from fish on days 0, 6 and 14. RNA was extracted from the tissue and evaluated on 1% agarose gel and Nanodrap. After cDNA synthesis, the samples were subjected to Real time PCR machine and gene expression analysis was performed using Sybr Green method. The results of this study indicated that the 500 ppm treatment stimulate vitellogenin gene expression at both 6^th and 14^th days exposure, when comparing with control (P< 0.05). After 6 day exposure, the fish treated with 200 ppm pulp and paper mill did not show significant difference in respect to vitellogenin gene expression, where comparing with control while significant increase (P< 0.05) was observed after 14^th day. This suggest pulp and paper mill  influent on vitellogenin gene expression and can be used as a biomarker for such wastewater.