Enzyme-linked immunosorbent assays (ELISAs) are tests that uses antibody recognition and enzyme catalytic activity to identify a substance, and they have been widely used as a diagnostic tool in the clinic. However, performing an ELISA requires various liquid handling steps and long binding times. To solve this problem, we developed a magnetic microfluidic ELISA system (MMF-ELISA). Integration with nickel magnetic nanoparticles can streamline the ELISA process in a fully automated manner forStreptococcus pneumoniaedetection. First, we synthesized paramagnetic surface-oxidized nickel nanoparticles (Ni/NiO NPs) to carry protein G. Then, we assembled a SUM290 (UlaG)-specific antibody on protein G. Finally, we integrated the NPs on a microfluidics chip forS. pneumoniaedetection. The chip contains three different layers to trap the solutions; the bottom layer SiO2is patterned on hydrophobic polymers and integrated with the middle layer PDMS and the top layer PMMA. With Arduino and motor IC, we developed an automated platform forS. pneumoniaedetection. Microfluidic ELISAs can reduce the manual handling and operation time. Furthermore, the developed system can be extended to multiple areas for ELISA-related assays. This economical, rapid and portable system may become a promising platform for sensingS. pneumoniaein clinical applications.