The invention discloses a semiconductor structure with a self-aligned spacer and a manufacturing method of the semiconductor structure with the self-aligned spacer. The manufacturing method includes providing a substrate, arranging at least two gate structures on the substrate, and exposing the position, between each two adjacent gate structures, of the substrate; forming a silicon oxide layer to cover the exposed substrate; forming a nitrogenous material to cover each gate structure and the silicon oxide layer; etching the nitrogenous material to form a sidewall of a first self-aligned spacer for each gate structure, and exposing part of the silicon oxide layer, wherein the sidewalls are opposite; removing the exposed silicon oxide layer to form a second self-aligned spacer, wherein a groove is defined by the first self-aligned spacer and the second self-aligned spacer; forming a contact plug in the groove.