Light quality affects plant leaf growth and its metabolism of amino acids and polyphenols. Anoectochilus roxburghii has been used for food, medicine, and ornamental purposes. To obtain the optimal light quality for improved accumulation of metabolites in A. roxburghii in vitro. The seedings were cultured under the white light (W), blue light (B), red light (R), red: blue light = 1:2 (R1B2), red: blue light = 1:1 (R1B1), red: blue light = 2:1 (R2B1). Twenty compounds in raw leaves were in situ rapidly identified within 20 s by neutral desorption-extractive electrospray ionization mass spectrometry (ND-EESI-MS) without any sample pretreatment, including eight amino acids, eleven polyphenols, and kinsenoside. Meanwhile, leaf area and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were evaluated. Our results showed that leaf area and antioxidant enzyme activities of the R2B1 were significantly higher than B or R group. The red-blue light increased the content of isoleucine, histidine, serine, arginine, and promoted the accumulation of caffeic acid, ferulic acid, quercetin, myricetin, kaempferol, and rutin in phenylpropane metabolism, especially in the R2B1, which could be used as the optimal light quality. ND-EESI-MS could enhance our understanding of the effect of light quality on the accumulation of amino acids and polyphenols in A. roxburghii.