Nano-formulations, as an innovative drug delivery system, offer distinct advantages in enhancing drug administration methods, improving bioavailability, promoting biodegradability, and enabling targeted delivery. By exploiting the unique size advantages of nano-formulations, therapeutic agents, including drugs, genes, and proteins, can be precisely reorganized at the microscale level. This modification not only facilitates the precise release of these agents but also significantly enhances their efficacy while minimizing adverse effects, thereby creating novel opportunities for treatment of a wide range of diseases. In this review, we discuss recent advancements, challenges, and future perspectives in nano-formulations for therapeutic applications. For this aim, we firstly introduce the development, design, synthesis, and action mechanisms of nano-formulations. Then, we summarize their applications in disease diagnosis and treatment, especially in fields of oncology, pulmonology, cardiology, endocrinology, dermatology, and ophthalmology. Furthermore, we address the challenges associated with the medical applications of nanomaterials, and provide an outlook on future directions based on these considerations. This review offers a comprehensive examination of the current applications and potential significance of nano-formulations in disease diagnosis and treatment, thereby contributing to the advancement of modern medical therapies.