Tightly　bound　extracellular　polymeric　substances　(TB-EPS)　are　important　components　of　sludge,　playing　a　crucial　role　in　the　behavior　of　activated　sludge.　They　are　located　in　the　innermost　layer　of　EPS　which　closely　combine　with　the　cell　surface　and　are　difficult　to　be　extracted.　To　fully　understand　the　role　of　TB-EPS,　it　is　extremely　important　to　find　an　appropriate　TB-EPS　extraction　protocol,　which　provides　maximum　yields　of　TB-EPS　under　the　premise　of　minimal　contribution　of　cell　lysis.　Ultrasonic　method　has　been　widely　applied　for　TB-EPS　extraction　due　to　its　unique　advantages,　but　no　one　has　developed　a　systematic　and　scientifically　optimized　method　for　this　protocol.　In　this　study,　a　novel　method　based　on　response　surface　methodology　(RSM)　was　successfully　developed　to　optimize　the　conditions　of　TB-EPS　extraction.　The　optimal　conditions　were　determined　at　an　ultrasound　time　of　1.00　min,　ultrasonic　density　of　5.59　W/mL　and　a　mixed　liquor　suspended　solid　(MLSS)　of　around　1700-1800　mg/L.　Furthermore,　combined　analysis　of　microscopy,　particle　size,　and　excitation-emission　matrix　(EEM)　was　successfully　applied　to　evaluate　the　optimal　conditions.　The　result　indicates　that　the　optimal　conditions　are　efficient,　reliable,　and　reproducible　which　effectively　solves　the　bottleneck　problem　of　the　conflict　between　cell　viability　and　TB-EPS　yield,　and　little　effect　on　its　chemical　structure.