AbstractAlthough a variety of urinary cancer markers are available forin vitrodiagnosis, inherent problems of urine environment—containing various inorganic/organic ions/molecules that vary in concentration over a 20‐fold range or more and significantly attenuate antibody avidity for markers—render conventional immunoassays unsuitable, remaining unresolved and a major challenge. Here we developed a 3D‐plus‐3D (3p3) immunoassay method, based on a single‐step urinary marker detection by 3D‐antibody probes, which are free of steric hindrance and capable of omnidirectional capture of markers in a 3D solution. The 3p3 immunoassay showed an excellent performance in the diagnosis of prostate cancer (PCa) through detecting PCa‐specific urinary engrailed‐2 protein, demonstrating 100% sensitivity and 100% specificity with the urine specimens of PCa‐related and other related disease patients and healthy individuals. This innovative approach holds a great potential in opening up a novel clinical route for precisein vitrocancer diagnosis and also pushing urine immunoassay closer to more widespread adoption.