•Microflow system based on cotton thread as the solution channel and screen-printed electrode as detector.•New voltammetric procedure for estriol determination in pharmaceutical formulations.•Development of electroanalytical thread-device of low cost and easy preparation.•Excellent analytical performance: low limit of detection, high sensitivity and stability of the proposed system. Microflow systems are powerful analytical tools that explore similar principles of typical flow injection analysis driven to in a microfluidic device. Generally, microfluidic devices can promote a low consumption of reagents and samples, high speed of analysis and possibility of portability. Several advances have been reached applying a simple and low cost device based on cotton thread as microfluidic channel where the transportation of solutions is based on capillary force helped by gravity. In the present work, we have demonstrated the versatility of thread-based electroanalytical devices (μTED) constructed using a cotton thread as the solution channel and screen-printed electrodes (SPE) surface modified with carbon nanotubes (CNT) as electrochemical detectors for the amperometric determination of estriol hormone in pharmaceutical samples. The parameters involved in the amperometric detection and microflow system were studied and optimized, using the best experimental conditions (flow rate of 0.50μLs−1, 10mm of analytical path, 2.0μL of volume of injection and potential of detection of 0.75V) a linear response was observed for concentration range (LDR) of 1.0 to 1000μmolL−1 with limits of detection (LOD) and quantification (LOQ) of 0.53μmolL−1 and 1.77μmolL−1, respectively, and frequency of injection of 32 per hour. The proposed methodology was applied for determination of estriol in commercial samples and results were compared with those provided by spectrophotometric method (official methodology). The obtained results are in agreement at a 95% of confidence level.