In this study, a novel wax-printed paper-based lateral flow device has been developed as an alternative approach for an automated and one-step enzyme-linked immunosorbent assay (ELISA). The design pattern consisted of a non-delayed channel, a wax-delayed channel, a test zone and a control zone. This system was easily fabricated on a nitrocellulose membrane using a wax-printing method and then baked in an oven at 100°C for 1min. The four barriers of the wax-delayed channel could delay the flow time for 11s compared to the flow time of the non-delayed channel. To use the device under optimal conditions, alpha-fetoprotein (AFP) was detected at a limit of detection of 1ngmL−1 and assessed with the naked eye within 10min. A colorimetric intensity was also measured using a smart phone and computer software at a linear range of 0.1–100ngmL−1 with a good correlation. Furthermore, the proposed device was successfully applied to detect AFP in human serum. Therefore, the wax-printing demonstrates a user-friendly, easy and quick method for the fabrication of the device, which could be used as a one-step, portable, disposable, low-cost, simple, instrument-free and point-of-care device for the automated ELISA. •A novel wax-printed paper-based lateral flow device was proposed as an automated and one-step ELISA.•A wax-printed pad consisted of non-delayed channel, wax-delayed channel, test and control zones.•The device provides fast point-of-care testing of AFP in human serum (10min).