AbstractJapanese encephalitis (JE), a neglected tropical zoonotic disease prevalent in south‐east Asian and western pacific countries, caused by the flavivirus JE virus (JEV), has a dearth of electrochemical point‐of‐care (PoC) diagnostic tools available to manage endemic breakouts. To overcome this, we have developed a screen‐printed carbon electrode (SPCE) immunosensor for rapid PoC detection of JEV nonstructural 1 (NS1) antigen (Ag), found circulating in serum of infected individuals using a smartphone based portable “Sensit” device. The modification of SPCE surface with JEV NS1 antibody (Ab) was confirmed via observation of globular protein structures via scanning electron microscopy (SEM), increase in electrode surface hydrophilicity via contact angle measurement and decrease in current via differential pulse voltammetry (DPV). The fabrication and testing parameters were optimized based on highest current output obtained using DPV. The SPCE was tested for detection limit of target JEV NS1 Ag ranging from 1 fM to 1 μM, which was determined as 0.45 fM in spiked serum. The disposable immunosensor was also found to be highly specific in detecting JEV NS1 Ag over other flaviviral NS1 Ag. Finally, the modified SPCE was clinically validated by testing 62 clinical JEV samples using both a portable miniaturized electrochemical “Sensit” device coupled with a smartphone and a laboratory‐based potentiostat. The results were corroborated with gold‐standard RT‐PCR and showed 96.77% accuracy, 96.15% sensitivity, and 97.22% specificity. Hence, this technique may further be developed into a one‐step rapid diagnostic tool for JEV, especially in rural areas.