As　one　of　the　great　advances　in　modern　technology,　the　microarray　is　widely　used　in　many　fields,　including　biomedical　research,　clinical　diagnosis,　and　so　on.　Evidently,　in　order　to　extract　the　intensity　of　fluorescence　bio-probes　accurately,　we　need　to　pay　special　attention　to　the　gridding　of　microarray　at　first.　To　solve　the　poor　effect　of　the　traditional　Otsu　method　for　microarray　gridding,　an　innovative　algorithm　of　Otsu　optimized　by　multilevel　thresholds　is　proposed　to　improve　the　accuracy　and　effectiveness　of　the　microarray　image　gridding　and　segmentation.　The　experimental　results　indicate　that　considering　the　physical　information　carried　by　microarrays,　the　improved　algorithm　of　Otsu　optimized　by　multilevel　thresholds　achieves　high-quality　gridding　and　establishes　the　bio-spot　coordinates　more　precisely.　Compared　with　the　traditional　Otsu　method,　its　gridding　error　is　reduced　to　zero,　and　the　integrated　relative　error　of　bio-spot　coordinates　is　decreased　from　2.89%　to　1.05%.　This　optimization　of　Otsu　combined　with　physical　information　of　spot-matrix　will　greatly　improve　the　performance　of　segmentation　so　as　to　make　the　contribution　to　extracting　the　fluorescence　intensity　of　microarray　accurately.　©　2013　IEEE.