Zearalenone (ZEN, ZEA) contamination in various foods and feeds is a significant global problem. Similar to deoxynivalenol (DON) and other mycotoxins, ZEN in feed mainly enters the body of animals through absorption in the small intestine, resulting in estrogen-like toxicity. In this study, the gene encoding Oxa, a ZEN-degrading enzyme isolated fromAcinetobacterSM04, was cloned intoLactobacillus acidophilusATCC4356, a parthenogenic anaerobic gut probiotic, and the 38 kDa sized Oxa protein was expressed to detoxify ZEN intestinally. The transformed strainL. acidophiluspMG-Oxa acquired the capacity to degrade ZEN, with a degradation rate of 42.95% at 12 h (initial amount: 20 μg/mL). The probiotic properties ofL. acidophiluspMG-Oxa (e.g., acid tolerance, bile salt tolerance, and adhesion properties) were not affected by the insertion and intracellular expression of Oxa. Considering the low amount of Oxa expressed byL. acidophiluspMG-Oxa and the damage to enzyme activity by digestive juices, Oxa was immobilized with 3.5% sodium alginate, 3.0% chitosan, and 0.2 M CaCl2to improve the ZEN degradation efficiency (from 42.95% to 48.65%) and protect it from digestive juices. The activity of immobilized Oxa was 32–41% higher than that of the free crude enzyme at different temperatures (20–80 °C), pH values (2.0–12.0), storage conditions (4 °C and 25 °C), and gastrointestinal simulated digestion conditions. Accordingly, immobilized Oxa could be resistant to adverse environmental conditions. Owing to the colonization, efficient degradation performance, and probiotic functionality ofL. acidophilus, it is an ideal host for detoxifying residual ZEN in vivo, demonstrating great potential for application in the feed industry.