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Non-destructive detection of the pH value of kiwifruit has important practical significance for its quality classification. In this study, hyperspectral fluorescence imaging technology was proposed to quantitatively predict the pH value of kiwifruit non-destructively. Firstly, the SPXY algorithm was used to divide samples into training and prediction sets and three different algorithms were used to preprocess the raw spectral data. Secondly, algorithms such as the iteratively retaining information variables (IRIV), the variable iterative space shrinkage approach (VISSA), the model adaptive space shrinkage (MASS), the random frog (RF), and their combination (i.e., IRIV + VISSA + MASS + RF, IVMR) were used to extract effective variables from the preprocessed spectral data. Moreover, the second extractions, such as IRIV-VISSA and IRIV-MASS, and the third extraction (i.e., IVMR-VISSA-IRIV) were used to further reduce the redundant variables. Based on the effective variables, four regression models—random forest (RF), partial least square (PLSR), extreme learning machines (ELM), and multiple-kernel support vector regression (MK-SVR)—were built and compared for predicting. The results show that IVMR-VISSA-IRIV-MK-SVR had the best prediction results, with <i>R_P</i>², <i>R_C</i>² and <i>RPD</i> of 0.8512, 0.8580, and 2.66, respectively, which verifies that hyperspectral fluorescence imaging technology is reliable for predicting the pH value of kiwifruit non-destructively.