The　objective　of　this　study　was　to　investigate　the　effects　of　culture　conditions,　including　carbon　sources　and　concentration,　culture　period,　and　precondition　time,　on　the　production　of　extracellular　polymeric　substances　(EPS)　and　its　influence　on　microalgal　flocculation.　EPS　are　natural　high　molecule　polymer,　excreted　by　microalgae　themselves.　EPS　can　accelerate　the　formation　of　microbial　aggregates　through　binding　cells　closely.　Organic　carbon　sources,　such　as　glucose,　glycerol,　acetate　and　glycine　were　compared　to　select　the　optimal　source　to　stimulate　EPS　accumulation.　Subsequently,　the　effect　of　culture　period,　glycine　dose　and　precondition　time　on　EPS　production　and　its　influence　on　biomass　growth　and　flocculation　efficiency　were　investigated.　As　the　main　parts　of　EPS,　tightly　bound　EPS　were　found　positively　related　to　suspended　solids　concentration.　However,　the　loosely　bound　EPS　may　weaken　the　floc　structure,　leading　to　poor　water-cells　separation.　Under　the　optimal　condition　with　culture　period　of　16　days,　glycine　dose　of　0.5　g　l-1　and　precondition　time　of　5　days,　the　biomass　concentration　increased　from　1.49　to　2　g　l-1,　and　the　maximum　suspended　solids　concentration　of　7.06%　with　biomass　recovery　rate　of　70.6%　was　achieved.　©　2015　The　Society　for　Biotechnology,　Japan.