Microfluidic paper-based analytical devices (μPADs) are promising biosensors that may be used in a variety of bioanalytical applications. A μPAD for automating the competitive enzyme-linked immunosorbent assay (ELISA) of small-sized target detection at the femtogram level using submicroliter samples is reported in this study. The proposed μPAD was integrated with a sucrose valve to automate the sequential delivery of reagents, providing simple control of reagent delivery time and simple operation. The use of a sample solution dropping location at the zones on the device that had been prepared with an antibody-conjugated enzyme before immersion in a running buffer allowed minimization of sample volume to 0.6 μL, while eliminating the possible loss of a target molecule by adsorption on the membrane, thus improving detection sensitivity. Furthermore, the proposed device was successfully applied to the automation of competitive ELISA for the detection of aflatoxin B1 (AFB1), a potent carcinogen that causes substantial health risks to humans worldwide, with a detection limit of 60 femtograms or 0.1 ng/mL. The method developed in this study provides high sensitivity, small sample volume, on-site and equipment-free measurements, low-cost operation, and user-friendliness. This approach could be used to analyze small-sized molecules in the fields of food safety and quality control, environmental monitoring, and clinical diagnostics.