Abstract: After being obtained through PCR amplification, glucoamylase gene promoter and tryptophan hydroxylase gene terminaer were linked together in sequence to construct Aspergillus niger secretory expression vector pPHT. EGFP gene was added to pPHT, forming a recombination expression vector, pPHT-EGFP.pPHT-EGFP was transformed into A.niger protoplasts. Positive transformants were identified through hygromycin resistence and PCR amplification. Positive transformants were cultured in a medium where starch was the sole carbon source. The hyphae were observed under a fluorescence microscope, indicating the characteristic of fluorescence distribution, which mainly concentrated on hyphae tips, septum, cell wall and culture medium. Green fluorescence of the solid medium indicated secretion of EGFP to extracellular medium，which belonged to secretory expression. Green fluorescence of other spaces showed positions of secretion.