Quasi-2D Ruddlesden-Popper perovskites receive tremendous attention for application in light-emitting diodes (LEDs). However, the role of organic ammonium spacers on perovskite film has not been fully-understood. Herein, a spacer cation assisted perovskite nucleation and growth strategy, where guanidinium (GA) spacer is introduced into the perovskite precursor and at the interface between the hole transport layer (HTL) and the perovskite, to achieve dense and uniform perovskite films with enhanced optical and electrical performance is developed. A thin GABr interface pre-formed on HTL provides more nucleation sites for perovskite crystal; while the added GA in perovskite reduces the crystallization rate due to strong hydrogen bonding interacts with intermediates, which promotes the growth of enhanced-quality quasi-2D perovskite films. The ionized ammonium group (-NH) of GA also favors formation of polydisperse domain distribution, and amine or imine (-NH or -NH) group interact with perovskite defects through coordination bonding. The spacer cation assisted nucleation and growth strategy is advantageous for producing efficient and high-luminance perovskite LEDs, with a peak external quantum efficiency of over 20% and a luminance up to 100 000 cd m. This work can inform and underpin future development of high-performance perovskite LEDs with concurrent high efficiency and brightness.