Rottenness is most prevalent and devastating disease that threats citrus fruit. Automatic detection of early rottenness can enhance the competitiveness and profitability of the citrus industry. However, there is no efficient automatic detection technology at this time that could detect this disease. The navel orange was selected as research objective. Hyperspectral fluorescence imaging was used to detect early rottenness in orange. Optimum index factor (OIF) method was applied to identify the optimal band combination. 100% detection rate was achieved based on the optimal bands ratio image and two threshold values. The research showed that the proposed method can effectively overcome the affect from florescence effect because stem damage area and stem also can produce florescence under ultraviolet light. This study will lay a foundation for developing multispectral detection system used in on-line detection of early rottenness fruit.