is an important industrial workhorse for the biomanufacturing of organic acids, proteins, etc. Well-controlled genetic regulatory elements, including promoters, are vital for strain engineering, but available strong promoters for are limited. Herein, to efficiently assess promoters, we developed an accurate and intuitive fluorescent-auxotrophic selection workflow based on , , CRISPR/Cas9 system, and flow cytometry. With this workflow, we characterized six endogenous constitutive promoters in . The endogenous glyceraldehyde-3-phosphate dehydrogenase promoter P showed a 2.28-fold increase in promoter activity compared with the most frequently used strong promoter P from . Six predicted conserved motifs, including the -box, were verified to be essential for the P activity. To demonstrate its application, the promoter P was used for enhancing the expression of citrate exporter in a citric acid-producing isolate D353.8. Compared with the controlled by P, the transcription level of the gene driven by P increased by 2.19-fold, which is consistent with the promoter activity assessment. Moreover, following overexpression, several genes involved in carbohydrate transport and metabolism were synergically upregulated, resulting in up to a 2.48-fold increase in citric acid titer compared with that of the parent strain. This study provides an intuitive workflow to speed up the quantitative evaluation of promoters and strong constitutive promoters for fungal cell factory construction and strain engineering.