The most common macrolide veterinary drugs used in animal husbandry are tylosin and tilmicosin. To protect human health, tylosin and tilmicosin residues in animal products and environmental water samples must be monitored on-site. A monoclonal antibody and gold nanoparticle-based lateral flow immunochromatographic strip (LFIS) were used in this study to detect tylosin and tilmicosin simultaneously, allowing for quantitative detection with a portable reader. The four-parameter equation fit produced a standard sigmoid curve under optimized conditions, with IC50 of 17.69 ng/mL and LOD(IC10) of 1.72 ng/mL for tylosin and IC50 of 18.95 ng/mL and LOD(IC10) of 7.07 ng/mL for tilmicosin. Additionally, good linearity results were obtained in the actual concentration range of 3.25–50 ng/mL, with R squares of 0.9946 for tilmicosin and 0.9953 for tylosin. The degree of cross-reactivity with other analogs was less than 0.1%. The average recoveries for tylosin and tilmicosin ranged from 77.5% to 119.7% and from 80% to 126.1% in milk, from 81.1% to 108.0% and from 98.3% to 106.8% in environmental water, respectively. The results could be obtained in 5–8 min. Therefore, the developed lateral flow immunochromatographic strip was met for the rapid detection of tylosin and tilmicosin residues in milk and environmental water samples using group screening. Furthermore, we discovered that the high matching of three dimensional structure at the distal structural fragments of the hapten arm site, as well as the consistency of the frontier orbital distribution, are very critical factors for the analytical performance of antibody against target molecules and should be considered as a universal theoretical guide for hapten design.