With the aim to investigate the physiological mechanism of tobacco response to molybdenum stress, the effect of photosynthetic pigment, gas exchange parameters，chlorophyll fluorescence parameters and chloroplast ultrastructure of tobacco were studied under molybdenum deficiency stress by the hydroponic experiment. The results showed that, compared with the control group, the photosynthetic pigment contents and net photosynthetic rate of tobacco leaves cultivated about 10d decreased， while intercellular CO2 concentration, stomatal conductance and transpiration rate increased. And the initial fluorescence and non-photochemical quenching coefficient (NPQ) of PS II increased, while photosynthetic electron transport rate (ETR) and the maximal photochemical efficiency decreased，but the differences between two treatments were not significant. The grana layer of the chloroplast ultrastructure under molybdenum deficiency stress expanded to deformation and loose，the number of amyloid was less and it grew worse. The most indicators between molybdenum deficiency and control tobacco cultivated about 20d were significant. In addition to Tr, other indexes between molybdenum deficiency and control tobacco cultivated about 30d were significant， and the chloroplast under the molybdenum deficiency stress was damaged seriously. Therefore，under the molybdenum deficiency stress，the chloroplast ultrastructure grows worse, the activity of PS II is inhibited, and the Pn decreases. With the extension of molybdenum stress time, the deficiency reaction is more obvious.