Kesterite-structured Cu2ZnSnSe4 (CZTSe) is considered as one of the Earth-abundant and non-toxic photovoltaic materials. CZTSe films have been prepared using a single-step co-evaporation method at a relatively low temperature (i.e., below 500 °C). Due to the volatile nature of tin-selenide, the control over substrate temperature (i.e., growth temperature) is very important in terms of the deposition of high-quality CZTSe films. In this regard, the effects of growth temperatures on the CZTSe film morphology were investigated. The suitable temperature range to deposit CZTSe films with Cu-poor and Zn-rich compositions was 380–480 °C. As the temperature increased, the surface roughness of the CZTSe film decreased, which could improve p/n junction properties and associated device performances. Particularly, according to capacitance-voltage (C-V) and derived-level capacitance profiling (DLCP) measurements, the density of interfacial defects of CZTSe film grown at 480 °C showed the lowest value, of the order of ~3 × 1015 cm−3. Regardless of applied growth temperatures, the formation of a MoSe2 layer was rarely observed, since the growth temperature was not high enough to have a reaction between Mo back contact layers and CZTSe absorber layers. As a result, the photovoltaic (PV) device with CZTSe film grown at 480 °C yielded the best power conversion efficiency of 6.47%. It is evident that the control over film growth temperature is a critical factor for obtaining high-quality CZTSe film prepared by one-step process.