To investigate the impact of multi-strain koji production on the process and quality of Zhejiang Rose Vinegar， a study was conducted to optimize the multi-strain ratio using an orthogonal experiment. The two best-performing mixed strains were applied to the production of rose vinegar， and a quality evaluation was performed. The results indicated that two ratios with the highest amylase and protease activities were 1 ∶ 1 ∶ 1 ∶ 1 and 1 ∶ 1 ∶ 1 ∶ 2 of Monascus purpureus ∶ Aspergillus oryzae ∶ A. niger ∶ Rhizopus， which were prepared and subsequently applied to the vinegar brewing process. Fermentation tracking showed that the multi-strain fermentation process for rose vinegar was successfully completed， with the total acidity of the final products reaching 6.97 g/100 mL and 6.63 g/100 mL， respectively， representing a yield increase of 17.21% and 11.29% compared to the traditional method. Eight flavor-contributing organic acids， including acetic acid， lactic acid， malic acid， and citric acid， were detected in the two rose vinegar samples， with non-volatile acid contents of 56.04 mg/L and 57.85 mg/L， respectively， which were comparable to the 41.053 mg/L of traditional rose vinegar. A total of 44 volatile flavor compounds were identified using headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS)， with volatile flavor compound contents of 6 348.14 mg/L and 6 791.75 mg/L in the two groups， slightly higher than the 5 030.07 mg/L in traditional vinegar. Sensory evaluation revealed that rose vinegar brewed with multi-strain koji showed slight improvements in sweetness and ester aroma， with the 1 ∶ 1 ∶ 1 ∶ 2 multi-strain group achieving the highest sensory scores. This study provides a reference for the mechanized improvement of koji production in traditional rose vinegar manufacturing.