The cation-independent bioflocculant (59LF) extracted from Klebsiella sp. 59L was characterized. 59LF consisted of protein (4.8%) and total sugar (85.2%) with high molecular weight (93.82% of 2120 kDa), and total sugar was composed of 76.45% of neutral sugar, 3.65% of uronic acid, and 1.43% of amino sugar. Results indicated that 59LF was pH tolerant and thermally stable, and the maximum yield of 59LF was 4.078 g/L after 48 h culture. The optimal flocculating activity for Kaolin particles was obtained when the dosage of 59LF was 7.0 mg/L without additional metal ions as coagulant aids. Furthermore, the surface properties of 59LF were observed using a Fourier-transform infrared spectrophotometer and X-ray photoelectron spectroscopy, whereas a porous structure was detected by a scanning electron microscope. Thus, a primary flocculation mechanism of 59LF was proposed. This study provided a potential cation-independent bioflocculant with high productivity and low dosage in future application.