Abstract: In order to investigate the effect of molecular crowding on the antifungal ability of DNA-AgNCs and its preservation effect on citrus, DNA sequence (5'-CCCCCCCCCCCC-3') was mixed with AgNO₃ solution in this experiment. DNA-AgNCs was prepared by simulating molecular crowded environment with 50% Polyethylene Glycol (PEG) 200 solution. Taking DNA-AgNCs as the research object, using Penicillium expansum, Aspergillus flavus, Fusarium graminaerum and Fusarium moniliforme as the models, the inhibition rate of DNA-AgNCs on the germination of four kinds of spores was determined by spore counting method, and the Minimal Inhibitory Concentration (MIC) of DNA-AgNCs against four kinds of fungi was determined by microdilution method. DNA-AgNCs was added to starch-chitosan matrix to prepare composite films, and its effects on the mechanical properties, hydrophobicity and antibacterial properties of the films were explored through tensile test, water contact angle and inhibition zone test. The film was applied to citrus preservation in the form of coating film, and the weight loss rate, plaque diameter and sensory evaluation of citrus were recorded. The results showed that the inhibitory effect of molecular crowded DNA-AgNCs on spore germination was better than that of normal DNA-AgNCs (P<0.05), and the MIC was within 2.5 μmol/L. The tensile properties of the films with molecular crowded DNA-AgNCs increased from 4.729 to 7.300 MPa, the elongation at break increased from 51.399% to 77.287%, and the water contact angle increased from 59.700° to 66.717°. The inhibition zones of the four fungi were 23.969, 19.176, 16.822 and 13.349 cm, respectively. When citrus was soaked and coated in the solution supplemented with molecular crowded DNA-AgNCs and stored at 28 ℃ for 14 d, the weight loss rate (23.8%) and plaque diameter (2.3 cm) were significantly lower than those of the control group (P<0.05), and the sensory evaluation (52 points) was significantly higher than that of the control group (P<0.05). In conclusion, molecular crowding could enhance the antibacterial ability of DNA-AgNCs, and the coating film could well maintain the storage quality of citrus.