The invention discloses a self-aggregation short-peptide induction based immobilized nitrilase and a preparation method and application thereof. The preparation method comprises the following steps: (1) splicing connecting peptides and self-aggregation short peptides, so as to construct an expression vector; (2) connecting a nitrilase gene and the expression vector, and transforming into a recipient strain, so as to obtain an engineering strain; (3) carrying out induced expression on the engineering strain, carrying out wall breaking treatment on cells, and centrifuging and/or filtrating to obtain a precipitate, namely a nitrilase endoenzyme aggregate; (4) immobilizing the obtained nitrilase endoenzyme aggregate in alginate, thereby obtaining the immobilized nitrilase. According to the immobilized nitrilase aggregate obtained by the method, the particle diameter of the immobilized nitrilase is about 1mm. According to the immobilized nitrilase, particles can be subjected to freeze-drying preservation, and the enzyme activity is reserved to 90% when the immobilized nitrilase is preserved for 20 days at the temperature of 37 DEG C; the activity of the immobilized nitrilase is high, and the operating flow is simple.