The invention provides a construction method of a mouse model with conditional mTERT overexpression and an application thereof. The construction method comprises the following steps: constructing a targeting vector through a seamless cloning technology, wherein the targeting vector comprises a 3.3 kb 5'homologous arm, a CAG promoter, loxp-stop-loxp, TERT-3XFlag-IRES-EGFP-WPRE-polyA and a 3.3 kb 3'homologous arm; cas9 mRNA, gRNA and the targeting vector are microinjected into fertilized eggs of wild type mice to obtain F0-generation mice, homologous recombination positive F0-generation mice are identified, F1-generation mice are obtained through breeding after backcross, and the F1-generation mice are conditional mTERT overexpressed mouse models. The mouse constructed by the construction method provided by the invention can mate with different Cre tool mice to realize specific overexpression in various tissues, organs and cell types of the whole body. The mouse can be used for modeling various transgenic models for researching embryo and nerve development, senescence, wound repair, tumor occurrence, development and treatment, and the mating of the mouse and a CreERT tool mouse can control the time of TERT overexpression through tamoxifen.