A new carbon paste electrode for rifampicin (RIF) drug was prepared and fully characterized in terms of composition, usable pH range and temperature. The sensor is based on 2-hydroxypropyl β-cyclodextrin as a good ionophore in the carbon paste matrix. The modified electrode showed a Nernstian slope of 59.2 mV/decade over the concentration range of 3.2 × 10−8 to 2.2 × 10−4 M with a limit of detection 2.3 × 10−8 M. The electrode has a short and stable response time of 4 s. The sensor manifested advantages of simple design, low cost, wide concentration range, excellent selectivity to rifampicin, applicable as an indicator electrode and renewability. The sensor was successfully used for determination of rifampicin in tablet and blood serum samples. Temperature dependence of the sensor potential response was examined in the temperature range of 15–55 °C. The sensor showed a very low thermal coefficient within the studied temperature range.