Background Approximately 30-36% of gliomas presented with [F-18]fluoroethyl-l-tyrosine ([F-18]FET) PET-negative at primary diagnosis, which interferes with the differentiation of gliomas from other isolated brain lesions. Preoperative noninvasive identification of [F-18]FET PET-negative gliomas to aggressive surgical treatment could reduce ineffective treatment and improve prognosis. This study aimed to assess the potential utility of multiparametric MRI with H-1-magnetic resonance spectroscopy (H-1-MRS) in the diagnosis of gliomas within [F-18]FET PET-negative isolated cerebral lesions. Results A total of 51 patients (mean age 44.35 +/- 27.15 years, 26 males) with 37 gliomas and 14 non-gliomas were recruited for the study. More than half of PET-negative gliomas presented T2-FLAIR mismatch sign, whereas non-gliomas were more likely to present absence of T2-FLAIR mismatch sign (54.05% vs. 7.14%, p < 0.001). Choline to creatine (Cho/Cr) ratios in gliomas were significantly higher than those in non-gliomas (2.21 vs. 1.30, p < 0.001). Multiparametric MRI (AUC = 0.88) outperformed conventional MRI (AUC = 0.72) in differentiating gliomas from non-gliomas (NRI = 0.29, p = 0.02). And WHO grade was correlated with Cho/Cr and total lesion tracer standardized uptake (TLU) (r = 0.43 and 0.55; p = 0.007 and < 0.001; respectively). Low-grade PET-negative gliomas exhibit low levels of both TLU and Cho/Cr, but the distribution of TLU and Cho/Cr is more variable in high-grade gliomas. Furthermore, there was a moderated correlation between TLU and Cho/Cr in low-grade PET-negative gliomas (r = 0.54, p = 0.017), whereas there was no correlation in the high-grade PET-negative gliomas (r = -0.017, p = 0.95). Conclusion Multiparametric MRI with H-1-MRS demonstrates significant promise in enhancing the diagnosis and overall clinical management for [F-18]FET PET-negative gliomas. Moreover, the correlation between TLU and Cho/Cr that was affected by tumor grading of 2021 WHO criteria provides a rationale for further research into the mechanisms of reduced [F-18]FET uptake in gliomas.