In this paper, we report the fabrication of a dual microsensor for sensing nitric oxide (NO) and calcium ions (Ca2+) and its application for simultaneous NO/Ca2+ measurements in living rat kidney tissue. NO and Ca2+ have very important physiological functions and are both intricately involved in many biological processes. The dual NO/Ca2+ sensor is prepared based on a dual recessed electrode possessing Pt (diameter, 25 μm) and Ag (diameter, 76 μm) microdisks. The Pt disk surface (WE1) is electrodeposited with porous Pt black and then coated with fluorinated xerogel; and used for amperometric sensing of NO. The Ag disk surface (WE2) is chloridated to AgCl, followed by silanization and then Ca2+ selective membrane loading; and used for potentiometric sensing of Ca2+. The dual sensor exhibits high sensitivity of WE1 to NO (40.8 ± 6.5 pA μM-1, n = 10) and reliable Nerntian response of WE2 to Ca2+ changes (25.7 ± 0.5 mV pCa-1, n = 10) with excellent selectivity to only NO and Ca2+ over common interferents and reliable stability (up to ∼4 h tissue experiment). The prepared sensor is employed for real-time monitoring of the dynamic changes of NO and Ca2+ levels of a rat kidney, which is induced by the administration of 10 mM l-NG-nitroarginine methyl ester (l-NAME, a NO synthase inhibitor). Due to the small sensor dimension, location-dependent analyses of NO and Ca2+ are carried out at two different regions of a kidney (renal medulla and cortex). Higher NO and Ca2+ levels are observed at the medulla than at the cortex. This study verifies the feasibility for real-time monitoring of intimately connected Ca2+ and endogenous NO production; and also for localized concentration assessments of both NO and Ca2+.