Alpine meadows on the Tibetan Plateau have experienced severe degradation in recent decades. Although the effects of alpine meadow degradation on soil properties have been well documented, there is still a paucity of knowledge regarding the responses of nitrogen-cycling microbes (NCMs) to degradation and their links to the changes in soil properties. Here, we systematically determined the effects of degraded patch formation on soil properties (i.e., total carbon, total nitrogen, ammonium nitrogen, nitrate nitrogen, available phosphorus, dissolved organic carbon, moisture, δ15N, δ13C, and pH) and NCMs (based on nifH, amoA, narG, nirK, and nirS genes and their transcripts) across three Tibetan alpine meadows at different degradation stages. Results showed that compared to the original grassed patches, the contents of most soil nutrients (e.g., carbon, nitrogen, and phosphorus) were significantly decreased in the degraded patches across the study sites. Degraded patches also tended to have higher soil δ15N values and nitrate contents. Among the aforementioned NCMs, soil diazotrophs and denitrifiers only showed weak responses to the patch formation, while ammonia-oxidizing microbes showed the highest consistency and sensitivity in response to the patch formation across the study sites. The abundance of amoA gene and archaeal amoA mRNA significantly increased in the degraded patches, and they were positively correlated with soil δ15N values and nitrate nitrogen contents, but negatively correlated with soil total nitrogen and inorganic nitrogen contents. These results suggest that the increased ammonia-oxidizing microbial abundance may be an important driver of soil nitrogen loss during degraded patch formation in alpine meadows.