Understanding the effect of light on stomatal behavior is mandatory for plant cultivation under controlled environmental systems. In this study, the effects of LED light quality and intensity on daily stomatal conductance (gs), stomatal aperture movement and stomatal density were investigated in petunia (Petunia hybrida Vilm.) plants under a semi-closed system. Three petunia cultivars, namely 'Coral', 'Purple' and 'Red', were grown under different light quality and quantity for 1 month and then all the parameters were measured. Results showed that 'Coral' exhibited overall higher gs than 'Purple' and 'Red'. Light quality significantly affected gs, width of stomatal aperture and stomatal density of all cultivars. The effect of light quality on gs and width of stomata aperture was strongly depended upon cultivar and time of day and there was a two-way interaction. Light intensity did not affect daily fluctuation pattern of gs but did affect the amplitude of the fluctuation. In contrast to the effect of light quality, the effect of light intensity was independent of cultivar and time of day. There was no obvious correlation between gs and width of either adaxial or abaxial stomata apertures during photoperiod. Light intensity significantly affected stomatal density which could partially explain an increasing of gs in response to increasing light intensity.