The invention relates to a chitosan nanofiber composite film for adsorbing and separating heavy metal ions and a preparation method and application thereof. The composite film is a chitosan electrospun nanofiber/non-woven fabric base composite film. The method for preparing the composite film comprises the following steps: performing electro-spinning on chitosan on a non-woven fabric base through an electrostatic spinning method, thereby obtaining a composite fiber film; and combining multiple composite fiber film layers in different manners, thereby preparing an adsorption and separation medium. The adsorption and separation medium is filled in a filter separator and can be used for dynamically adsorbing and filtering to remove heavy metal ions in water. The composite film disclosed by the invention is simple and convenient in preparation method, green, environmental-friendly, low in cost, easy to popularize and apply, high in heavy metal wastewater treatment efficiency and high in selectivity, can be used for purifying and treating heavy metals in industrial and domestic wastewater, and has important social and economical benefits.