The immobilizing fermentation characteristics and bioflocculant production of two bioflocculant-producing bacteria, Agrobacterium tumefaciens F2 and Bacillus sphaeicus F6 using mycelial pellets as biomass carrier were investigated. The optimal parameters of fermentation for the best flocculation efficiency were determined via response surface methodology (RSM) as follows: seed age 24 h, inoculum amount 3.6% (m/m), initial pH 7.7, temperature 29.5 °C, and rotation speed 140 rpm. Under these optimal conditions, combined mycelial pellets were transferred repeatedly for over 30 cycles for bioflocculant production. The bacterial biomass, flocculating efficiency and the bioflocculant yield were stable during the semi-continuous production process. Bioflocculant produced after 18–30 h fermentation had the highest yield. Scanning electron microscope images showed that the bioflocculant-producing bacteria were immobilized on the mycelial pellets. Using mycelial pellets as a biomass carrier for the semi-continuous fermentation of bioflocculant is feasible and promising for future industrial production applications.