Stimuli-responsive self-assembly of functional amphiphilic molecules by specific chemical stimulants is a promising strategy for sensor application. Herein, we demonstrate a fast optical detection of urea in human urine by exploiting bolaform perylene diimide functionalized with imidazoles (PDI-Hm), whose aggregation is induced by urea hydrolysis. The hydroxides produced from the enzymatic urea hydrolysis deprotonate the imidazoles to reduce electrostatic repulsion between PDI-Hm molecules in a HCl-methanol mixture, thereby leading to aggregation and consequent fluorescence quenching. The molecular interaction of PDI-Hm was further scrutinized to understand the aggregation behavior driven by the screening of electrical repulsion. As an optical sensing probe, PDI-Hm displays a prompt response (<1 min) to hydroxide and detection limit of 0.4 mM for urea. PDI-Hm incorporating urease offers considerable selectivity toward urea among various components in human urine. The urea sensing accuracy of this PDI-Hm fluorescence chemosensor is comparable to that of a clinical method, showing 93.4% consistency. Furthermore, the PDI-Hm was fabricated into a gel film allowed for the fast screening of excessive urea in urine.