A non-volatile memory and a manufacturing method thereof are provided. The non-volatile memory includes a substrate, a gate structure, a first doped region, a second doped region and a pair of isolation structures. The gate structure is disposed on the substrate. The gate structure includes a charge storage structure, a gate and spacers. The charge storage structure is disposed on the substrate. The gate is disposed on the charge storage structure. The spacers are disposed on the sidewalls of the gate and the charge storage structure. The first doped region and the second doped region are respectively disposed in the substrate at two sides of the charge storage structure and at least located under the spacers. The isolation structures are respectively disposed in the substrate at two sides of the gate structure.