The incorporation of biopolymers and natural colorants in smart packaging has garnered significant attention in the food packaging industry. This study investigates the design and characterization of novel fibrous films incorporating betalain extract (BE) from Stenocereus thurberi in poly (lactic acid) (PLA). An electrospinning technique was developed with varying PLA concentrations (2%–12% w/v) and BE concentrations (8%–12% w/v) to create a colorimetric freshness indicator. BE was characterized by quantifying its phytochemical content and assessing its antioxidant capacity. Morphological and structural analyses included scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), polydispersity index (PI), mechanical properties, and functional characteristics such as ammonia sensitivity and total antioxidant activity. The results indicated that the incorporation of BE significantly influenced the average diameter of the nanofibers, ranging from 313 ± 74 nm to 657 ± 99 nm. SEM micrographs showed that PLA12-BE12 films exhibited smooth surfaces without bead formation. The FTIR analysis confirmed effective BE incorporation, revealing intermolecular interactions between the betalain molecules and the PLA matrix, which contributed to enhanced structural and functional stability. The mechanical properties analysis revealed that moderate BE additions (8%–10% w/v) enhanced the Young’s modulus and tensile strength, while higher BE concentrations (12% w/v) disrupted the polymer network, reducing these properties. Additionally, the strain at break decreased significantly with BE incorporation, reflecting limited molecular chain mobility. Increasing BE concentration notably improved antioxidant activity, with the BE concentration of 12% (w/v), the ABTS•+, DPPH•, and FRAP radical scavenging activities at the highest values of 84.28 ± 1.59%, 29.95 ± 0.34%, and 710.57 ± 28.90 µM ET/g, respectively. Ammonia sensitivity tests demonstrated a significant halochromic transition from reddish-pink to yellow, indicating high sensitivity to low ammonia concentrations. The possible mechanism is alkaline pH induces aldimine bond hydrolysis and generates betalamic acid (yellow) and cyclo-DOPA-5-O-ß-glucoside (colorless) The fibrous films also exhibited reversible color changes and maintained good color stability over 30 days, emphasizing their potential for use in smart packaging applications for real-time freshness monitoring and food quality assessment.