Escherichia coli strain Nissle 1917 (EcN) is one kind of probiotics and has been shown to colonize preferentially in tumor tissues. Electrospun fibers have been created for a localized drug delivery to alleviate the systemic toxicity and promote the drug accumulation in tumors. Up to now no attempt has been made to encapsulate genetically engineered bacteria in electrospun fibers for drug delivery purposes. In the current study, plasmids containing reporter genes encoding green fluorescent protein (GFP) were constructed, followed by transformation into EcN by electroporation. The engineered bacteria were entrapped into the cores of coaxially electrospun fibers of poly(ethylene glycol)-polylactide (PELA), and 600 nm-sized NaCl microparticles were blended in matrix polymers to create diffusion paths in the fiber sheath for mass changes. Up to 48% of bacteria entrapped within fiber cores were alive at around 5 × 105 cfu mg-1 fibers, and the entrapped bacteria proliferated in the confined environment of fiber cores with a passage time of around 12 h. Additionally, the engineered bacteria were grafted on the fiber surface via covalent linking by glutaraldehyde or affinity adsorption on mannose-grafted fibers. The affinity absorption of bacteria led to a significantly higher immobilization efficiency than that after covalent binding, at 2 × 105 and 8 × 104 cfu mg-1 fibers, respectively. GFP expressions were determined in the culture media for EcN bacteria after either entrapment or grafting on fibers. This study provides a strategy to immobilize engineered bacteria on electrospun fibers for efficient gene delivery and disease management.