Research and development of methods to assess fish freshness continues to be a major challenge for the fishing industry. At the same time, consumers are now increasingly attentive to food quality, including fish freshness and product history. Here, we propose a reliable, rapid and easy-to-apply fluorimetric approach to assess fish freshness using a micro-volume fluorimeter. Mitochondrial functions were assessed in European seabass (Dicentrarchus labrax) fillets at different durations of storage at +4 °C: from Day 0 to Day 8. We found that mitochondrial respiration and mitochondrial membrane potential (ΔΨm) were significantly disrupted after 4 days of storage at +4 °C. The spectral properties (emission peak and fluorescence intensity) of mitochondrial membrane potential probes rhodamine 123 (Rh123) and tetramethylrhodamine methyl ester (TMRM) were strongly affected by the ΔΨm integrity of the fish fillets. We highlighted two categories of fish quality as a function of ΔΨm: Day 0 to Day 3, ΔΨm was preserved; and D4 to D7, ΔΨm was disrupted. Thus, evaluation of ΔΨm constitutes an early and reliable predictive indicator of fish freshness.