Micro-sized　glassy　carbon　microspheres　(GCMs,　typically　3　mu　m　in　diameter)　instead　of　nano-sized　gold　nanoparticles　(AuNPs,　typically　20　nm　in　diameter)　were　for　the　first　time　used　as　signal　markers　for　the　quantitative　detection　of　antigen　such　as　prostate-specific　antigen　(PSA).　After　being　treated　with　concentrated　HNO3,　GCMs　bear　carboxyl　groups　at　their　surfaces,　which　enables　antibodies　to　be　conjugated　with　GCMs　to　yield　new　type　of　micro-sized　material-based　colorimetric　probes　used　for　immunochromatographic　test　strips　(ICTSs).　The　captured　black　GCMs　(with　strong　and　wide-band　light　absorption)　on　the　T-line　of　ICTS　were　used　both　for　qualitative　and　quantitative　determination　of　PSA.　In　the　case　of　quantitative　determination,　a　lab-assembled　optical　strip　reader　system　was　used　to　measure　the　reflected　LED　light　intensity　at　550　nm.　The　sensing　performances　of　the　developed　GCM-based　ICTSs,　such　as　sensitivity,　selectivity,　reproducibility,　stability,　and　applicability,　were　investigated　in　detail.　The　developed　GCM-based　ICTSs　can　have　much　higher　(3　times)　detection　sensitivity　than　AuNP-based　ICTSs,　showing　promising　applications　in　sensitive　immunoassay.