Identification of differentially expressed genes (DEGs) in time course studies is very useful for understanding gene function, and can help determine key genes during specific stages of plant development. A few existing methods focus on the detection of DEGs within a single biological group, enabling to study temporal changes in gene expression. To utilize a rapidly increasing amount of single-group time-series expression data, we propose a two-step method that integrates the temporal characteristics of time-series data to obtain a B-spline curve fit. Firstly, a flat gene filter based on the Ljung–Box test is used to filter out flat genes. Then, a B-spline model is used to identify DEGs. For use in biological experiments, these DEGs should be screened, to determine their biological importance. To identify high-confidence promising DEGs for specific biological processes, we propose a novel gene prioritization approach based on the partner evaluation principle. This novel gene prioritization approach utilizes existing co-expression information to rank DEGs that are likely to be involved in a specific biological process/condition. The proposed method is validated on the Arabidopsis thaliana seed germination dataset and on the rice anther development expression dataset.