The instant disclosure relates to a method for forming self-aligned isolation trenches in semiconductor substrate, comprising the following steps. The first step is providing a semiconductor substrate defined a plurality of active areas thereon. The next step is forming at least two buried bit lines in each of the active areas and an insulating structure disposed above and opposite to the at least two buried bit lines. The next step is forming a self-aligned spacer on the sidewalls of each of the insulating structures. The last step is selectively removing the semiconductor substrate with the self-aligned spacers as masks to form a plurality of isolation trenches.