The　objective　of　the　study　was　to　investigate　the　change　of　biofilm　characteristics　when　implementing　the　procedure　of　partial　nitrification.　A　ratio　control　strategy　(DO/NH4+-N)　was　taken　to　achieve　partial　nitrification,　and　biofilm　samples　were　obtained　at　10.27%,　52.12%,　and　93.54%　of　the　nitrite　accumulation　rate.　The　amount　and　spatial　distribution　of　total　bacteria,　ammonia　oxidizing　bacteria　(AOB),　and　nitrite　oxidative　bacteria　(NOB)　were　observed　by　fluorescence　in　situ　hybridization　(FISH)　and　confocal　laser　scanning　microscope　(CLSM)　through　a　three-dimensional　excitation　emission　matrix　(EEM)　to　observe　the　secretion　and　composition　changes　of　extracellular　polymer　substances.　Ratio　control　successfully　enriched　AOB　and　achieved　partial　nitrification　under　conditions　when　NOB　was　not　completely　washed.　Heterotrophic　bacteria　and　nitrifying　bacteria　coexist　in　the　biofilm.　The　heterotrophic　bacteria　were　in　the　outer　layer,　but　nitrifying　bacteria　were　distributed　in　the　biofilm　surface　at　6-25　μm.　During　the　process　of　short-range　nitrification,　the　AOB/NOB　value　gradually　increased,　and　the　stable　operation　period　was　as　high　as　15.56.　During　the　operation　of　the　reactor,　EPS　and　microbial　flora　changes　are　closely　related.　When　microbial　activity　decreased,　EPS　secretion　decreased.　During　the　stable　operation　period　of　partial　nitrification,　NOB　and　other　bacteria　that　are　non-resistant　to　high　nitrite　nitrous　acid　declined,　and　the　fluorescence　intensity　of　aromatic　protein-like　bacteria　decreased.　However,　the　three-dimensional　fluorescence　spectra　showed　that　the　chemical　composition　of　EPS　was　not　obvious　during　the　process　of　partial　nitrification.　©　2018,　Science　Press.　All　right　reserved.