Novel constitutive promoters are essential for plant biotechnology. Although in angiosperms, a number of promoters were applied in monocots or dicots genetic engineering, only a few promoters were used in gymnosperm. Here we identified two strong promoters ( and ) from Chinese fir () by screening the transcriptomic data and preliminary promoter activity assays in tobacco. By using the newly established Chinese fir protoplast transient expression technology that enables molecular biology studies in its homologous system, we compared the activities of and with that of the commonly used promoters in genetic engineering of monocots or dicots, such as , , and , and our results revealed that and promoters have stronger activities in Chinese fir protoplasts. Furthermore, the vector containing gene driven by promoter and sgRNA driven by the newly isolated promoters were introduced into Chinese fir protoplasts, and CRISPR/Cas mediated gene knock-out event was successfully achieved. More importantly, compared with the commonly used promoters in the genetic engineering in angiosperms, promoter has much stronger activity than promoter in transgenic poplar, and promoter in transgenic rice, respectively, indicating its potential application in poplar and rice genetic engineering. Overall, the novel putative constitutive gene promoters reported here will have great potential application in gymnosperm and angiosperm biotechnology, and the transient gene expression system established here will serve as a useful tool for the molecular and genetic analyses of Chinese fir genes.