限制酶（英语：restriction enzyme）又称限制内切酶或限制性内切酶（restriction endonuclease），全称限制性核酸内切酶，是一种能将双股DNA切开的酶。切割方法是将糖类分子与磷酸之间的键结切断，进而于两条DNA链上各产生一个切口，且不破坏核苷酸与碱基。切割形式有两种，分别是可产生具有突出单股DNA的黏状末端，以及末端平整无凸起的平滑末端。。由于断开的DNA片段可由另一种称为DNA连接酶的酵素黏合，因此染色体或DNA上不同的限制片段，得以经由剪接作用而结合在一起。

A restriction enzyme or restriction endonuclease is an enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites. Restriction enzymes are commonly classified into three types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.