Objective To survey genome-scale protease profiles regulated by the Aspergillus niger transcription factor PrtT and further controlled by carbon sources. Results The PrtT disruption mutant (delprtT) and overexpression (OEprtT) strains were successfully generated and further confirmed by phenotypic and protease activity analysis. RNA-seq analysis of WT and mutants identified 32 differentially expressed protease genes, which mostly belonged to serine-type peptidases, aspartic-type endopeptidases, aminopeptidases and carboxypeptidases. Furthermore, based on the MEME predicted motif analysis of the PrtT promoter, EMSA and phenotypic and qRT-PCR analyses confirmed that the carbon metabolism regulator AmyR directly regulated the protease genes and their regulatory factor PrtT. Conclusion Thirty-two PrtT-regulated protease genes were identified by RNA-seq, and the secondary carbon source regulator AmyR was found to have a negative regulatory effect on the expression of PrtT and its target protease genes.