Mice are important laboratory animals used in biological and medical studies. Pathogen infections in laboratory mice are detected using the enzyme-linked immunosorbent assay (ELISA), which requires a large quantity of serum. A low serum requirement is important for more frequent and detailed analyses for infections. A new serology monitoring system with a 16-microchannel microfluidic chip was developed to identify anti-pathogen antibodies in 0.2 mu L of mouse serum; the system indicates infections caused by 6 important murine pathogens: Salmonella enterica serotype Typhimurium, Sendai virus, mouse hepatitis virus, Mycoplasma pulmonis, lymphocytic choriomeningitis virus, and ectromelia virus. To avoid non-specific adsorption of mouse sera to the microchannel, a polyvinyl alcohol-based blocking method was developed. To equalize the sensitivities of different antigens, the quantities of pathogen antigens deposited were optimized through precision spotting by using the electrospray deposition method. A comparative study with the conventional ELISA method was carried out, and the developed microfluidic chip system was able to simultaneously and specifically detect antibodies against the above mentioned 6 pathogens with good linearity (R-2 = 0.955-0.966) in 12 min with a lower serum quantity (1/13), reaction time (1/16), and antigen quantities (1/19-1/2,800) than the conventional ELISA. Validation using a serum panel (n = 501) also showed good agreement with previous results.