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Luteolin from <i>Patrinia villosa</i> exhibits strong antiviral activity. Here, the conditions for extracting and enriching luteolin from <i>P. villosa</i> were optimized. Response surface methodology was used to determine the optimal extraction parameters in terms of reflux time, solvent ratio, extraction temperature, material-to-liquid ratio, and number of extractions. Thereafter, a macroporous resin method was used to enrich luteolin from <i>P. villosa</i>. Finally, the following optimal extraction and enrichment conditions were established: an extraction time of 43.00 min, a methanol/hydrochloric acid solvent ratio of 13:1, an extraction temperature of 77.60 °C, a material/liquid ratio of 1:22, and a total of two extractions. NKA-9 was determined to be the most appropriate resin for enrichment. The ideal adsorption conditions were as follows: a pH of 5.0, a temperature of 25 °C, an initial luteolin concentration of 19.58 µg/mL, a sample loading volume of 2.9 BV, and a sample loading rate of 2 BV/h. The ideal desorption conditions were as follows: distilled water, 30% ethanol and 80% ethanol elution, and 5 BV at a flow rate of 2 BV/h. After optimization, the enrichment recovery rate was 80.06% and the luteolin content increased 3.8-fold. Additionally, the enriched product exhibited a significant inhibitory effect on PRV (Porcine pseudorabies virus) in vitro and in vivo, providing data for developing and applying luteolin from <i>P. villosa</i>.