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Objective: Glioblastoma multiforme is a highly aggressive form of brain cancer, and early diagnosis plays a pivotal role in improving patient survival rates. In this regard, molecular magnetic resonance imaging has emerged as a promising imaging modality due to its exceptional sensitivity to minute tissue changes and the ability to penetrate deep into the brain. This study aimed to assess the efficacy of a novel contrast agent in detecting gliomas during MRI scans., Materials and Methods: The contrast agent utilized modified chitosan coating on manganese oxide nanoparticles. The modification included adding methotrexate and 5-aminolevulinic acid (MnO 2 /CS@5-ALA-MTX) to target cells with overexpressed folate receptors and breaking down excess hydrogen peroxide in tumor tissue, resulting in enhanced signal intensity in T 1 -weighted MR images but diminished signal intensity in T 2 *-weighted MR images., Results: The nanosystem was characterized and evaluated in MR imaging, safety, and ability to target cells both in vivo and in vitro. MTX-free nanoparticles (MnO 2 /CS@5-ALA NPs) had no obvious cytotoxicity on cell lines U87MG and NIH3T3 after 24/48 h at a concentration of up to 160 µgr/mL (cell viability more than 80%). In this system, methotrexate enables tumor targeting and the MnO 2 /5-ALA improves T 1 -T 2 * -weighted MRI. In addition, MRI scans of mice with M109 carcinoma indicated significant tumor uptake and NP capacity to improve the positive contrast effect., Conclusion: This developed MnO 2 /CS@5-ALA-MTX nanoparticle system may exhibit great potential in the accurate diagnosis of folate receptor over-expressing cancers such as glioblastoma., (© 2024. The Author(s), under exclusive licence to European Society for Magnetic Resonance in Medicine and Biology (ESMRMB).)