Recently,numerous biological macromolecular experiments have been conducted with optical tweezers.For the single molecular stretching experiment with optical tweezers,three ways to determine the initial adhesion point of DNA on the coverslip are described in this work.In addition,a new method through analyzing the displacement variance of the trapped particle to obtain the trap height is introduced.Using our proposed methods,the obtained force-extension curve for the operated dsDNA agrees well with the worm-like chain model.These improved methods are also applicable to other related biological macromolecular experiments requiring high precision.