The timing of onsets is a critical factor in preventing disability in stroking patients. Developing a faster and more accurate diagnostic method is essential. Copeptin, as a stable surrogate for arginine vasopressin, has emerged as a novel and reliable serum biomarker for stroke. Therefore, to facilitate prompt stroke detection, this work attempts to develop a quick and precise technique for identifying copeptin in human serum. For homogenous analysis, a novel amplified luminescence method based on aldehyde was created. This method employs the double antibody sandwich technique to detect copeptin levels in human serum. Serum samples from stroke patients, individuals with stroke-like symptoms but no confirmed stroke, and healthy controls were analyzed and compared. The detection time was shortened to 10 min. Stroke patients exhibited significantly higher copeptin concentrations compared to patients with stroke-like symptoms but no confirmed stroke diagnosis. Furthermore, both groups of patients demonstrated significantly elevated copeptin concentrations relative to healthy subjects. The combined diagnostic approach using copeptin and homocysteine (Hcy) markedly enhanced diagnostic efficiency. Copeptin-AlphaLISA, an amplified luminous proximity homogenous assay, was successfully developed. Serum copeptin demonstrates significant potential in the rapid and combined diagnosis of stroke.