The composition of the medium, particularly the nutrition elements and plant growth regulators, plays a crucial role in in vitro shoot proliferation. Knowing the effect of each of these factors and their interaction effects on in vitro growth traits requires multiple and complex experiments. In this study, we conducted an multi-factor experiment to examine the effects of five medium components (NH4NO3, KNO3, mesos, micros, iron, and BAP) at various concentrations on the in vitro shoot proliferation of Vitis labrusca 'Concord' using a response surface model with 42 runs. The findings of this study suggest that KNO3 and BAP exerted the most significant positive and negative effects on the proliferated shoot number, respectively. A decrease in NH4NO3 concentration was found to promote an increase in shoot length. Moreover, KNO3 and NH4NO3 were observed to negatively regulate both leaf number and leaf color, highlighting their influence on these morphological traits. Our results indicate that the precise amount and form of nitrogen sources play a critical role in the in vitro shoot production. The interaction between BAP and KNO3, iron, and other micros had a significant impact on shoot proliferation traits. Specifically, utilizing reduced concentrations of NH4NO3 and KNO3 (in range 0.5-2.5× MS medium), along with 2.25 mg.l-1 BAP, is recommended for achieving enhanced shoot production, shoot length, leaf number, and leaf color in Vitis labrusca 'Concord'. These findings provide a valuable foundation for further research pertaining to in vitro studies in Vitis species.