Objective The exosome, a type of extracellular vesicle, is considered a kind of information carrier and biomarker and has a significant role in extracellular physiologic activities and disease progression. Research into the characteristics of exosomes is key to addressing numerous fundamental issues in pathologic processes. Exosomes are secreted by tumor cells into the tumor microenvironment through exocytosis. Exosome vesicles carry a large amount of genetic information related to the tumor cells from which they are derived, which plays an important role in the tumor microenvironment, such as information transmission, immune regulation, promotion of tumor invasion and metastasis, and regulation of tumor response to drugs. At present, most exosomes research focuses on the bioinformatics exploration of protein expression, mRNA, lncRNA and other aspects of exosomes, and there is still a lot of space to explore the microscopic morphology and physical characteristics of exosomes. This study investigated the physical characteristics of plasma exosomes from patients with lymphoma through atomic force microscopys. Methods The exosome has been extracted from clinical cancer patients’ peripheral blood samples. The samples contain diffuse large B cell lymphoma and follicular lymphoma. At first, the electrostatic adsorption method has been utilized to fix the isolated exosomes onto the mica substrates, the discrete living exosomes have been scanned with AFM method. The multi-parameter exosome images at nano/micro scale can be obtained in situ. Results The multi-parameter mechanical properties of isolated living exosomes have been visualized and quantitatively measured. The significant fringe effect at the edge of the exosome has been revealed in the AFM adhesion image, which means more energy dissipation can be produced there. The young's modulus of plasma exosomes was different in patients with lymphoma from different pathological subtypes. Conclusion Plasma exosomes from patients with hematological tumors can be successfully extracted. Due to the convenient and less invasive characteristics of peripheral blood, plasma exosomes can be used as biomarkers for dynamic monitoring and analysis of tumor diagnosis and treatment, and the extracted exosomes can be captured by AFM probes through treatment and fixation. In this study, the morphological details and mechanical properties of exosomes at nano and micro scales were revealed. The physical properties of exosomes may be related to their biological properties. The multi-dimensional understanding of exosome performance opens up a new perspective for the future application and in-depth study of exosomes.