Lateral flow assay, a simple and rapid test to detect various agents; have found many applications in different fields. One of the most important issues in the construction of a Lateral Flow assay is the increasing of its sensitivity and efficiency by processing of analytical layer. This study, aimed to investigate the performance of, Nellulose, Nitrocellulose, Nylon and PVD as different analytical layer in morphine Lateral flow strip as a model. Cyanogens bromide, acetonitrile, methanol and specific polymers were used to activate cellulose, nitrocellulose, PVDF and nylon respectively. BSA-morphine and anti morphine polyclonal antibodies were immobilized orderly in test and control bands different concentrations of morphine were prepared as sample solution. Results showed that activated supports have better detection level in comparison with control strips. Among activated supports nitrocellulose showed more reliable results rather than others and introduced as a suitable support for construction of analytical layer and antibody immobilization. Nylon and PVDF supports due to its hydrophobic nature and cellulose due to high capillary property and non-uniform texture showed inappropriate results. Using proper supports for design of lateral flow strips can improve the sensitivity and detection level of this system.