Aluminum (Al) toxicity limits crop yield in acidic soil through affecting diverse metabolic processes, especially photosynthesis. The aim of this work was to examine the effect of Al on photosynthetic electron transport in vivo as determined by chlorophyll fluorescence and delayed fluorescence of tobacco leaves. Results showed that Al treatment inhibited the photosynthetic rate and electron transfer, and decreased photosystem (PS) II photochemical activity in a time- and concentration-dependent manner, which could not be obviously alleviated by the addition of the reactive oxygen species (ROS) scavenger ascorbic acid (AsA). These results suggested that photosynthetic electron transfer chain components, especially PSII, might be directly damaged by Al instead of in an ROS-dependent manner. Furthermore, the fluorescence imaging and biochemical analysis exhibited that Al, after entering the cells, could accumulate in the chloroplasts, which paralleled the decreased content of Fe in the chloroplast. The changes in the chlorophyll fluorescence decay curve, the delayed fluorescence decay curve and the chlorophyll fluorescence parameters indicated that Al, through interacting with or replacing the non-heme iron between Q(A) and Q(B), caused the inhibition of electron transfer between Q(A) and Q(B), resulting in PSII photochemical damage and inhibition of the photosynthetic rate. In summary, our results characterized the target site of Al phytotoxicity in photosynthetic electron transport, providing new insight into the mechanism of Al phytotoxicity-induced chloroplast dysfunction and photosynthetic damage.