The invention provides neoisoquinoline alkaloid separated from red clover as well as a preparation method and application thereof. The preparation method comprises the following steps: grinding driedred clover, soaking in ethanol, heating and performing reflux extraction, and drying the filtrate in a low-temperature environment until no alcohol taste exists so as to obtain total thick paste of the red clover; uniformly dispersing the total thick paste of the red clover into a HCl solution, performing ultrasonic treatment, standing and filtering to obtain the supernatant; extracting the supernatant with an organic solvent to obtain thick paste; eluting with a petroleum ether-ethyl acetate elution system, a cyclohexane-ethyl acetate elution system and methanol in sequence, and finally repeatedly eluting with acetonitrile, thereby obtaining the compound. The compound is novel in structure and simple and convenient in preparation method, has an obvious anti-platelet aggregation effect, and has high medicinal value.