Abstract: Broccoli is highly susceptible to postharvest black spot disease caused by Alternaria brassicicola. However, the early-stage biomarkers and metabolic indicators of this disease remain unclear. In this study, the changes in weight loss, relative conductivity, color parameters, yellowing index, chlorophyll, total phenolics, flavonoids, and volatile organic compounds (VOCs) in broccoli inoculated with A. brassicicola and in control samples were systematically analyzed under storage at room temperature (25±1 ℃) for 4 days and low temperature (4±1 ℃) for 40 days. The aim was to identify key biomarkers and metabolic indicators associated with black spot disease. The results showed that during storage at room temperature, the flavonoid content in the first day inoculation group was significantly higher than that in the control group (P<0.01), the weight loss rate and a^* of the second day inoculation group were significantly higher than those of the control group (P<0.05), while the L^* and b^* of the inoculation group were significantly lower than those of the control group (P<0.01). There was no significant difference in chlorophyll content between the two groups, the content of hexanal and trans-2-hexenal in the control group showed an initial increase followed by a decrease, while the content of hexanal and trans-2-hexenal in the inoculation group showed a continuous decrease trend. During low-temperature storage, the weight loss rate, L^*, and chlorophyll content of the 10th day inoculation group were significantly lower than those of the control group (P<0.05), while the total phenolic content of the inoculation group was significantly higher than that of the control group (P<0.01), indicating that phenolic substances accumulate in response to infection at low temperatures. On the 20th day of low-temperature storage, the a^* of the inoculation group was significantly higher than that of the control group, and the b^* of the inoculation group was significantly lower than that of the control group (P<0.05). The content of hexanal and trans-2-hexenal showed a trend of first decreasing, then increasing, and finally decreasing. Furthermore, broccoli inoculated with A. brassicicola under room temperature conditions exhibited significant weight loss and increased cell membrane permeability. In conclusion, the study confirmed that weight loss and color parameters are critical biomarkers for black spot disease in broccoli caused by A. brassicicola, and VOCs, particularly trans-2-hexenal and hexanal, serve as key indicators of early infection. Additionally, The changes in total phenolic and chlorophyll content at low temperatures can also be used as indicators for disease identification. These findings provide theoretical support for the rapid diagnosis of postharvest black spot disease in broccoli.