This cross-sectional study examined data from the National Health and Nutrition Examination Survey (NHANES) conducted between 2007 and 2016, ultimately including 12,442 participants to explore the association between serum Klotho levels and asthma prevalence. IBL ELISA method, while asthma diagnosis relied on participant responses from NHANES questionnaires. A weighted multifactorial logistic regression analysis evaluated the relationship, and a smoothing curve assessed potential nonlinearity .After adjusting for confounding variables, a significant inverse association was found between serum Klotho concentrations and asthma incidence. Specifically, each unit increase in log-transformed Klotho reduced asthma risk by 48% (OR:0.52;95% CI:0.38–0.69). The analysis also revealed that participants in the lowest Klotho quartile had a higher prevalence of asthma compared to those in the highest quartile (OR:0.58;95% CI:0.45–0.76).Subgroup analyses indicated interactions related to drinking status and a history of cardiovascular disease. Notably, the smoothing curve did not identify a non-linear relationship. These findings suggest that lower serum Klotho levels are significantly associated with higher asthma prevalence in middle-aged and elderly Americans, highlighting the need for further cohort studies to clarify the causal relationship between these variables.