This study reported the production of pH-responsive films based on 8 wt% polyvinyl alcohol solution/0.2 wt% agar solution incorporated with cochineal-loaded starch particles (CSN) (2, 4, 6 and 8 wt% on agar basis) by a casting process. Results revealed that CSN presented obvious color changes over the pH range of 2–12. FTIR, XRD spectra and SEM micrographs presented that the incorporation of CSN formed new hydrogen bonds with a matrix and a tighter network structure. A certain improvement was observed in the color stability, swelling index and functional properties (antimicrobial and antioxidant activities) but water solubility, water vapor permeability and water contact angle of the pH-responsive films were decreased by the addition of CSN. The release of cochineal was a rate-limiting step following the Korsmeyer-Peppas model. The agar/polyvinyl alcohol film containing 6% CSN (PVA/GG-6) exhibited the best sensitivity for ammonia detection and its limit of detection was 35.4 ppm (part per million) for ammonia. The application trials showed that the PVA/GG-6 film presented different color changes for pork freshness. Hence, these pH-responsive films can be used as potential packaging materials for tracking the freshness of protein-rich fresh food in a non-destructive way.