In this work, a boronic acid copolymer, poly(4-vinylphenylboronic acid-co-2-(dimethylamino)ethyl methacrylate-co-n-butyl methacrylate) (pVDB) was designed for the rapid detection of bacteria based on reversible boronate ester formation with the diol-rich saccharide moieties found on bacterial membranes. Electrospun nanofibre membranes were prepared from a blend of polyacrylonitrile (PAN) and pVDB, which was synthesized by free radical polymerization. The pVDB@PAN membranes were used as fluorescent bacterial biosensors, displaying a distinct emission at 538 nm when in contact with bacteria cells. The fluorescence intensity showed a maximum intensity after 24 h of contact with Staphyloccocus aureus or Escherichia con, and this intensity increased proportionally with the pVDB content in the electrospun membranes. When in contact with Pseudomona putida, the membranes became non-responsive within 8 h due to the rapid formation of a bacterial biofilm. This fouling phenomena blocked the membrane surface for fluorescence readings. The pVDB@PAN bionsensor allowed for the rapid detection of the early stages of bacterial colonization well before biofilm formation, which could be advantageous for the early identification of pathogenic bacteria and prevention of their spreading.