Despite combination antiretroviral therapy (cART), adolescents with perinatally acquired human immunodeficiency virus (PHIV) exhibit cerebral injury and cognitive impairment. Plasma neurofilament light (pNfL) is a biomarker identified as a promising marker associated with neuroaxonal injury and cognitive impairment. To investigate whether cerebral injury in cART-treated PHIV adolescents is persistent, we longitudinally measured pNfL. We included 21 PHIV adolescents and 23 controls, matched for age, sex, ethnic origin and socio-economic status. We measured pNfL in both groups and CSF NfL in PHIV adolescents using a highly sensitive Single Molecule Array (Simoa) immunoassay. We compared pNfL between groups over time with a mean followup time of 4.6 years and assessed its association with MRI outcomes, cognitive function and HIV-related characteristics using linear mixed models. The median age was 17.5 years (15.5–20.7) and 16.4 years (15.8–19.6) at the second assessment for PHIV adolescents and controls, respectively. We found comparable pNfL (PHIV vs. controls) at the first (2.9 pg/mL (IQR 2.0–3.8) and 3.0 pg/mL (IQR 2.3–3.5), p = 0.499) and second assessment (3.3 pg/mL (IQR 2.5–4.1) and 3.0 pg/mL (IQR 2.5–3.7), p = 0.658) and observed no longitudinal change (coefficient; −0.19, 95% −0.5 to 0.1, p = 0.244). No significant associations were found between pNfL and HIV-or cART-related variables, MRI outcomes or cognitive function. We observed low CSF NfL concentrations at the baseline in PHIV adolescents (100.8 pg/mL, SD = 47.5). Our results suggest that there is no ongoing neuroaxonal injury in cART-treated PHIV adolescents and that the neuroaxonal injury is acquired in the past, emphasizing the importance of early cART to mitigate HIV-related neuroaxonal damage.