Lucerne (Medicago sativa L.) is a major component of the crops used in dry-land farming systems in China and its management is associated with notable nitrous oxide (N2O) emissions. A high proportion of these emissions is more likely to occur during periods when the soil undergoes freezing and thawing cycles. In this study, the effects of freeze/thaw cycles on N2O emissions and related factors were investigated in lucerne grasslands. The hypothesis was tested whether increased emissions resulted from a disruption of nitrification or denitrification caused by variations in soil temperatures and water contents. Three days (3 × 24 h) were chosen, where conditions represented freezing and thawing cycles. N2O emissions were measured for a fallow control (F) and two grasslands where lucerne had been cultivated for 4 and 11 years. Soil temperature, soil water content, soil microbial biomass carbon (MBC), soil microbial biomass nitrogen (MBN), soil ammonium nitrogen (NH4+-N), and soil nitrate nitrogen (NO3−-N) contents were measured. Moreover, the quantities of soil nitrification and denitrification microbes were assessed. Variations in N2O emissions were strongly affected by freeze/thaw cycles, and emissions of 0.0287 ± 0.0009, 0.0230 ± 0.0019, and 0.3522 ± 0.0029 mg m−2 h−1 were found for fallow, 4-year-old, and 11-year-old grasslands, respectively. Pearson correlation analyses indicated that N2O emissions were significantly correlated with the soil water content, temperature, NH4+-N content, and the number of nitrosobacteria and denitrifying bacteria at a soil depth of 0–100 mm. The numbers of nitrosobacteria and denitrifying bacteria correlated significantly with soil temperature at this soil depth. MBN and soil NH4+-N contents correlated significantly with soil water content at this depth. Principal component analysis highlighted the positive effects of the number of denitrifying bacteria on N2O emissions during the freeze/thaw period. Furthermore, soil temperature and the number of nitrosobacteria at the tested soil depth (0−100 mm) also played a significant role. This shows that soil freeze/thaw cycles strongly impacted both N2O emissions and the diurnal range, and the number of denitrifying bacteria was mainly influenced by soil temperature and soil NH4+-N content. The number of denitrifying bacteria was the dominant variable affecting N2O emissions from lucerne grasslands during the assessed soil freeze/thaw period on the Loess Plateau, China.