Water soluble zwitterionic fluorescent conjugated boronic acid-bearing polyfluorene (PFBA) has been prepared from poly(9,9′-(6′′-bromohexyl)fluorene-co-alt-1,4-phenylene) (polymer 3) through a post-polymerized quaternization with 3-pyridineboronic acid. Titration of diol containing monosaccharides (D-glucose and D-fructose), L-ascorbic acid and L-DOPA with PFBA polymer in 0.1 M phosphate buffer (pH 7.4) solution results in significant concentration dependent quenching of the blue fluorescence of the polymer due to static quenching as the bio-analytes form a ground state boronate ester complex with PFBA. Upon addition of D-glucose, the emission colour of PFBA changes to greenish-yellow owing to the effective induced aggregated polymer structure. PFBA also exhibits maximum response to the biological analytes at pH 7.4 which provides a scope for PFBA to be used in biological systems.