Nanoplastics (NPs) pose significant ecological risks due to their small size and ability to penetrate biological tissues across all trophic levels. However, their biological effects remain incompletely understood, particularly in tropical reef ecosystems. This study investigated the effects of short-term (7-day) exposure to carboxyl-modified polystyrene nanoparticles (PS-NPs, 100 nm) on juvenile clownfish (Amphiprion ocellaris), using two concentrations: low (20 µg/L, environmentally relevant) and high (2000 µg/L). A multidisciplinary approach combining biochemical and transcriptomic analyses was used to assess toxicity. Biochemical assays revealed limited changes in oxidative stress biomarkers (CAT, GR, GST, and TOSCA assays). However, the Integrated Biomarker Response index (IBRv2i) suggested compromised fish health, a finding supported by transcriptomic data. Gene expression analysis showed that both concentrations induced significant changes, with shared differentially expressed genes (DEGs) mostly upregulated, indicating a core molecular response. Despite this, each treatment also elicited unique transcriptional signatures. GO enrichment analysis of the high-concentration group highlighted processes related to muscle contraction, extracellular matrix organization, and oxidative stress responses, suggesting structural and physiological remodeling. In contrast, the low-concentration group showed enrichment for sensory system development, particularly visual function, as well as neurodevelopment and metabolic processes. These results indicate that even low NP concentrations can trigger substantial molecular alterations, although the nature of the biological response varies by exposure level. While high exposure is associated with stress adaptation and tissue restructuring, low exposure predominantly affects neural and sensory pathways. In conclusion, this study demonstrates that PS-NP exposure can alter gene expression and physiological state in juvenile reef fish, even at environmentally relevant concentrations. The distinct molecular profiles observed at different concentrations underscore the importance of exposure level in determining affected biological systems and potential long-term impacts of nanoplastic pollution in marine environments.