Serum alpha-klotho (s-klotho) protein has been linked with lifespan, and low concentrations of s-klotho have been associated with worse physical and cognitive outcomes. Although its significance in aging remains unclear, s-klotho has been proposed as a molecular biomarker of frailty and dependence. This study is a secondary analysis of data from a clinical trial performed in a population of 103 older individuals living in 10 nursing homes in Gipuzkoa (Spain). We aimed to elucidate associations between s-klotho (as measured by enzyme-linked immunosorbent assay) and body composition, physical fitness, and cognition, as well as frailty and dependence (determined using validated tests and scales). In addition, we investigated the association of s-klotho concentration with falls in the six months following the initial assessment. Low s-klotho levels were associated with a lower score in the psychological component of the Tilburg Frailty Indicator, a worse score in the Coding Wechsler Adult Intelligence Scale, and a greater dependence in activities of daily living. Moreover, participants with lower s-klotho concentrations suffered more falls during the 6 months after the assessment. Future translational research should aim to validate klotho's putative role as a biomarker that could identify the risk of aging-related adverse events in clinical practice.