A lab-on-a-chip (LoC) was designed for simultaneous monitoring of microorganisms, antibiotic residues, somatic cells, and pH in raw milk. The LoC was fabricated from polydimethylsiloxane (PDMS) using microelectromechanical system (MEMS) technology, which consisted of two parts; a protein array and microchannel. The protein array was fabricated by immobilizing five types of antibodies corresponding to two microorganisms, two antibiotic residues, and somatic cells. A sol-gel film was deposited on a glass substrate to immobilize the antibodies. The target analytes in raw milk could be bound with the corresponding antibody by an immunoreaction, and the antigen-antibody complex was detected using fluorescence microscopy. SNARF-dextran was used as a pH indicator, and the SNARF-entrapped hydrogel was attached to the microchannel in the chip. After injecting the milk sample into the channel, the pH was measured by monitoring the change in fluorescence intensity by fluorescence microscopy. The on-chip simultaneous assay of two microorganisms (E. coli O157:H7 and Streptococcus agalactiae), two antibiotic residues (penicillin G and dihydrostreptomycin), and neutrophils was successfully accomplished using the proposed LoC system.