Prosthetic valve endocarditis is a serious complication after heart valve replacement, accounting for about 20-30% of infective endocarditis (IE). Aspergillosis infection accounts for 25-30% of fungal endocarditis, and the mortality rate is 42-68%. Aspergillus IE often has negative blood cultures and lacks fever, which makes diagnosis difficult and delays antifungal therapy. Our study reported a case of IE in a patient with Aspergillus infection after aortic valve replacement. Ultra-multiplex polymerase chain reaction was used to identify Aspergillus infection and guide treatment. The purpose of this study was to enhance the understanding of the management of patients with endocarditis infected by fungi after valve replacement regarding the early detection, timely intervention, and treatment of the fungal infection to reduce the risk of death and improve the long-term survival of patients.