A transparent, lateral-flow test strip coupled with a smartphone-based ambient light sensor was first proposed for detecting enzymatic inhibition and phosphorylation. The principle of the platform is based on the simultaneous measurement of the total amount of the enzyme and enzyme activity to biomonitor exposure to organophosphorus (OP) pesticides. In this study, butyrylcholinesterase (BChE) was adopted as the model enzyme and ethyl paraoxon was chosen as an analyte representing OP pesticides. The total amount of BChE was quantified by a sensitive colorimetric signal originating from a sandwich immunochromatographic assay utilizing PtPd nanoparticles as a colorimetric probe, which exhibited excellent catalytic activity for phenols. In the sandwich immunoassay, only one antibody against BChE was simultaneously utilized as the recognition antibody and the labeling antibody due to the tetrameric structure of native BChE. The BChE activity was estimated by another colorimetric signal using the Ellman assay. Both colorimetric signals on two separated test strips were detected by the smartphone-based ambient light sensor. The proposed sensor operated with an LED in a 3D-printed substrate, which emitted excitation light and transmitted it through a transparent, lateral-flow test strip. With the increase in the colorimetric signal in the test line of the test strip, the intensity of the transmitted light decreased. The smartphone-based sensor showed excellent linear responses for assaying the total amount of BChE and active BChE ranging from 0.05 to 6.4 nM and from 0.1 to 6.4 nM, respectively. A high portability and low detection limit were simultaneously realized in the common smartphone-based device. This low-cost, portable, easy-operation, and sensitive immunoassay strategy shows great potential for online detection of OP exposure and monitoring other disease biomarkers.