By using a Li-6400 portable photosynthesis system, the diurnal dynamics of the photosynthetic characteristics of three alfalfa varieties Medicago sativa L. cv. Aohan, M. sativa L. cv. Rangelander, and M. sativa L. cv. Algonquin were studied. The results showed that for the test alfalfa varieties, the diurnal variation curves of their P(n), T(r), and WUE were not all presented double-peak. For Aohan and Algonquin, the maximum P(n) and T(r) occurred at 9:00-11:00; while for Rangelander, they appeared at 11:00. The minimum P(n) and T(r) of the three varieties all appeared at 7:00 and 19:00. The maximum WUE of Rangelander and Algonquin appeared at 7:00 and that of Aohan was at 9:00, and the minimum WUE of the three varieties all appeared at 15:00-17:00. Comprehensive analysis on the photosynthetic performance indices P(n), T(r), and WUE indicated that 2 years old alfalfa was in the best state, followed by 1 year old, and 4 years old. Correlation analysis showed that air temperature (T(a)) was the main environmental factor affecting P(n) and T(r), followed by water vapor pressure deficit (VPD) and relative humidity (RH).