Positive affect (PA) is not only associated with individuals’ psychological and physical health, but also their cognitive processes. However, whether medial temporal lobe (MTL) and its subfields’ volume/functional connectivity can explain individual variability in PA remains understudied. We investigated the morphological (i.e., gray matter volume; GMV) and functional characteristics (i.e., resting-state functional connectivity; rsFC) of PA combined with univariate and multivariate pattern analyses (MVPA) using a large sample of participants (n = 321). We simultaneously collected the T1-weighted (n = 321), high-resolution MTL T2-weighted, and resting-state functional imaging data (n = 209). The MTL and its subfields’ volumes, including the CA1, CA2 + 3, DG, and subiculum (SUB), perirhinal cortex (PRC), and parahippcampus (PHC), were extracted by automatic segmentation of hippocampal subfields (ASHS) software. The morphological results revealed that GMVs in the prefrontal-occipital and limbic (i.e., hippocampus, amygdala, and PHC) systems were associated with variability in PA at the whole-brain level using MVPA but not univariate analysis. Linear regression results further revealed a positive association between the MTL subfields’ GMV, especially for the right PRC, and PA after controlling for several covariates. PRC-seed-based rsFC analyses further revealed that its couplings with the fronto-parietal-occipital system predicted PA in both univariate and MVPA. These findings provide novel insights into the neuroanatomical and functional substrates underlying human PA trait. Findings also suggest critical contributions of the MTL and its subfield of the perirhinal cortex, but not hippocampal subfields, as well as its functional coupling with the fronto-parietal control-system on the formation of PA.